Polypeptides, antibody variable domains and antagonists

ABSTRACT

The invention relates to anti-TNFR1 polypeptides and antibody single variable domains (dAbs) that are resistant to degradation by a protease, as well as antagonists comprising these. The polypeptides, dAbs and antagonists are useful for as therapeutics and/or prophylactics that are likely to encounter proteases when administered to a patient, for example for pulmonary administration, oral administration, delivery to the lung and delivery to the GI tract of a patient, as well as for treating inflammatory disease, such as arthritis or COPD.

RELATED APPLICATIONS

This application is a continuation of U.S. National Stage applicationSer. No. 12/663,502 filed Jun. 8, 2010 under 35 USC §371, ofInternational Application No. PCT/GB2008/050405, filed Jun. 4, 2008 andpublished in English which claims priority under 35 USC §119, or 35 USC§365, to U.S. Provisional Application No. 60/933,632 filed Jun. 6, 2007and United Kingdom, Application No. 0724331.4, filed Dec. 13, 2007. Theentire teachings of the above applications are incorporated herein byreference.

The present invention relates to protease resistant polypeptides,immunoglobulin (antibody) single variable domains and anti-TumorNecrosis Factor 1 (TNFR1, p55, CD120a, P60, TNF receptor superfamilymember 1A, TNFRSF1A) antagonists comprising these. The invention furtherrelates to uses, formulations, compositions and devices comprising suchanti-TNFR1 ligands.

BACKGROUND OF THE INVENTION

Polypeptides and peptides have become increasingly important agents in avariety of applications, including industrial applications and use asmedical, therapeutic and diagnostic agents. However, in certainphysiological states, such as inflammatory states (e.g., COPD) andcancer, the amount of proteases present in a tissue, organ or animal(e.g., in the lung, in or adjacent to a tumor) can increase. Thisincrease in proteases can result in accelerated degradation andinactivation of endogenous proteins and of therapeutic peptides,polypeptides and proteins that are administered to treat disease.Accordingly, some agents that have potential for in vivo use (e.g., usein treating, diagnosing or preventing disease) have only limitedefficacy because they are rapidly degraded and inactivated by proteases.

Protease resistant polypeptides provide several advantages. For example,protease resistant polypeptides remaining active in vivo longer thanprotease sensitive agents and, accordingly, remaining functional for aperiod of time that is sufficient to produce biological effects. A needexists for improved methods to select polypeptides that are resistant toprotease degradation and also have desirable biological activity.

TNFR1

TNFR1 is a transmembrane receptor containing an extracellular regionthat binds ligand and an intracellular domain that lacks intrinsicsignal transduction activity but can associate with signal transductionmolecules. The complex of TNFR1 with bound TNF contains three TNFR1chains and three TNF chains. (Banner et al., Cell, 73(3) 431-445(1993).) The TNF ligand is present as a trimer, which is bound by threeTNFR1 chains. (Id.) The three TNFR1 chains are clustered closelytogether in the receptor-ligand complex, and this clustering is aprerequisite to TNFR1-mediated signal transduction. In fact, multivalentagents that bind TNFR1, such as anti-TNFR1 antibodies, can induce TNFR1clustering and signal transduction in the absence of TNF and arecommonly used as TNFR1 agonists. (See, e.g., Belka et al., EMBO,14(6):1156-1165 (1995); Mandik-Nayak et al., J. Immunol, 167:1920-1928(2001).) Accordingly, multivalent agents that bind TNFR1, are generallynot effective antagonists of TNFR1 even if they block the binding ofTNFα to TNFR1.

The extracellular region of TNFR1 comprises a thirteen amino acidamino-terminal segment (amino acids 1-13 of human TNFR1; amino acids1-13 of mouse TNFR1), Domain 1 (amino acids 14-53 of human TNFR1; aminoacids 14-53 of mouse TNFR1), Domain 2 (amino acids 54-97 of human TNFR1;amino acids 54-97 of mouse TNFR1), Domain 3 (amino acids 98-138 of humanTNFR1; amino acid 98-138 of mouse TNFR1), and Domain 4 (amino acids139-167 human TNFR1; amino acids 139-167 of mouse TNFR1) which isfollowed by a membrane-proximal region (amino acids 168-182 of humanTNFR1; amino acids 168-183 mouse TNFR1). (See, Banner et al., Cell 73(3)431-445 (1993) and Loetscher et al., Cell 61(2) 351-359 (1990).) Domains2 and 3 make contact with bound ligand (TNFβ, TNFα). (Banner et al.,Cell, 73(3) 431-445 (1993).) The extracellular region of TNFR1 alsocontains a region referred to as the pre-ligand binding assembly domainor PLAD domain (amino acids 1-53 of human TNFR1; amino acids 1-53 ofmouse TNFR1) (The Government of the USA, WO 01/58953; Deng et al.,Nature Medicine, doi: 10.1038/nm1304 (2005)).

TNFR1 is shed from the surface of cells in vivo through a process thatincludes proteolysis of TNFR1 in Domain 4 or in the membrane-proximalregion (amino acids 168-182 of human TNFR1; amino acids 168-183 of mouseTNFR1), to produce a soluble form of TNFR1. Soluble TNFR1 retains thecapacity to bind TNFα, and thereby functions as an endogenous inhibitorof the activity of TNFα.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a polypeptide comprising an aminoacid sequence that is at least 93% identical to the amino acid sequenceof DOM1h-131-206 (shown in FIG. 3 and SEQ ID NO: 4). In one embodiment,the percent identity is at least 94, 95, 96, 97, 98 or 99%. In oneembodiment, the polypeptide is DOM1h-131-206. The invention furtherprovides (substantially) pure DOM1h-131-206 monomer. In one embodiment,the DOM1h-131-206 is at least 98, 99, 99.5% pure or 100% pure monomer.

In one aspect, the invention provides a polypeptide encoded by an aminoacid sequence that is at least 80% identical to the nucleotide sequenceof the nucleotide sequence of DOM1h-131-206 (shown in FIG. 19). In oneembodiment, the percent identity is at least 70, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides a polypeptide encoded by anucleotide sequence that is at least 57% identical to the nucleotidesequence of DOM1h-131-206 (shown in FIG. 19) and wherein the polypeptidecomprises an amino acid sequence that is at least 93% identical to theamino acid sequence of DOM1h-131-206 (shown in FIG. 3). In oneembodiment, the percent identity of the nucleotide sequence is at least60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. Inone embodiment, the percent identity of the amino acid sequence is atleast 94, 95, 96, 97, 98 or 99% or 100%. For example, the nucleotidesequence may be a codon-optimised version of the nucleotide sequence ofDOM1h-131-206 (shown in FIG. 19). Codon optimization of sequences isknown in the art. In one embodiment, the nucleotide sequence isoptimized for expression in a bacterial (e.g., E. coli or Pseudomonas,e.g. P. fluorescens), mammalian (e.g., CHO) or yeast host cell (e.g.Picchia or Saccharomyces, e.g. P. pastoris or S. cerevisiae).

In one aspect, the invention provides a fusion protein comprising thepolypeptide of the invention.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is at least 93% identical to the amino acid sequenceof DOM1h-131-206 (shown in FIG. 3). In one embodiment, the percentidentity is at least 94, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides a protease resistant anti-TNFαreceptor type 1 (TNFR1; p55) immunoglobulin single variable domaincomprising an amino acid sequence that is at least 90% identical to theamino acid sequence of DOM1h-131-206. In one embodiment of theseaspects, the percent identity is at least 80, 85, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%.

In one embodiment, the immunoglobulin single variable domain comprisesaspartic acid at position 53, wherein numbering is according to Kabat(“Sequences of Proteins of Immunological Interest”, US Department ofHealth and Human Services 1991).

In one embodiment, the immunoglobulin single variable domain compriseshistidine at position 91, wherein numbering is according to Kabat.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain encoded by anucleotide sequence that is at least 80% identical to the nucleotidesequence of DOM1h-131-206 (shown in FIG. 19). In one embodiment, thepercent identity is at least 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97,98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain encoded by anucleotide sequence that is at least 57% identical to the nucleotidesequence of DOM1h-131-206 (shown in FIG. 19) and wherein the variabledomain comprises an amino acid sequence that is at least 93% identicalto the amino acid sequence of DOM1h-131-206 (shown in FIG. 3). In oneembodiment, the percent identity of the nucleotide sequence is at least60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. Inone embodiment, the percent identity of the amino acid sequence is atleast 94, 95, 96, 97, 98 or 99% or 100%. For example, the nucleotidesequence may be a codon-optimised version of the nucleotide sequence ofDOM1h-131-206 (shown in FIG. 19). Codon optimization of sequences isknown in the art. In one embodiment, the nucleotide sequence isoptimized for expression in a bacterial (e.g., E. coli or Pseudomonas,e.g. P. fluorescens), mammalian (e.g., CHO) or yeast host cell (e.g.Picchia or Saccharomyces, e.g. P. pastoris or S. cerevisiae).

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain encoded by a sequencethat is identical to the nucleotide sequence of DOM1h-131-206 (shown inFIG. 19).

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist comprising an anti-TNFR1 immunoglobulin single variabledomain according to the invention. In one embodiment, the antagonistcomprises first and second immunoglobulin single variable domains,wherein each variable domain is according to invention. For example,wherein the antagonist comprises a monomer of said single variabledomain or a homodimer of said single variable domain. In one embodiment,the amino acid sequence of the or each single variable domain isidentical to the amino acid sequence of DOM1h-131-206 (shown in FIG. 3).

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more than 25 amino acid positions and has a CDR1sequence that is at least 50% identical to the CDR1 sequence ofDOM1h-131-206. In one embodiment, the difference is no more than 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 amino acid position. In one embodiment, the CDR sequence identityis at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more 25 than amino acid positions and has a CDR2sequence that is at least 50% identical to the CDR2 sequence ofDOM1h-131-206. In one embodiment, the difference is no more than 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 amino acid position. In one embodiment, the CDR sequence identityis at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more than 25 amino acid positions and has a CDR3sequence that is at least 50% identical to the CDR3 sequence ofDOM1h-131-206. In one embodiment, the difference is no more than 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 amino acid position. In one embodiment, the CDR sequence identityis at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more than 25 amino acid positions and has a CDR1sequence that is at least 50% identical to the CDR1 sequence ofDOM1h-131-206 and has a CDR2 sequence that is at least 50% identical tothe CDR2 sequence of DOM1h-131-206. In one embodiment, the difference isno more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position. In one embodiment, oneor both CDR sequence identities is respectively at least 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more than 25 amino acid positions and has a CDR1sequence that is at least 50% identical to the CDR1 sequence ofDOM1h-131-206 and has a CDR3 sequence that is at least 50% identical tothe CDR3 sequence of DOM1h-131-206. In one embodiment, the difference isno more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position. In one embodiment, oneor both CDR sequence identities is respectively at least 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more than 25 amino acid positions and has a CDR2sequence that is at least 50% identical to the CDR2 sequence ofDOM1h-131-206 and has a CDR3 sequence that is at least 50% identical tothe CDR3 sequence of DOM1h-131-206. In one embodiment, the difference isno more than 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid position. In one embodiment, oneor both CDR sequence identities is respectively at least 55, 60, 65, 70,75, 80, 85, 90, 95, 96, 97, 98 or 99%.

In one aspect, the invention provides an anti-TNFα receptor type 1(TNFR1; p55) immunoglobulin single variable domain comprising an aminoacid sequence that is identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3) or differs from the amino acid sequenceof DOM1h-131-206 at no more than 25 amino acid positions and has a CDR1sequence that is at least 50% identical to the CDR1 sequence ofDOM1h-131-206 and has a CDR2 sequence that is at least 50% identical tothe CDR2 sequence of DOM1h-131-206 and has a CDR3 sequence that is atleast 50% identical to the CDR3 sequence of DOM1h-131-206. In oneembodiment, In one embodiment, the difference is no more than 24, 23,22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2 or 1 amino acid position. In one embodiment, one or two or each CDRsequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90, 95, 96,97, 98 or 99% respectively.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR1 sequence that is at least 50% identical tothe CDR1 sequence of DOM1h-131-206 (shown in FIG. 3). In one embodiment,the CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR2 sequence that is at least 50% identical tothe CDR1 sequence of DOM1h-131-206 (shown in FIG. 3). In one embodiment,the CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR3 sequence that is at least 50% identical tothe CDR1 sequence of DOM1h-131-206 (shown in FIG. 3). In one embodiment,the CDR sequence identity is at least 55, 60, 65, 70, 75, 80, 85, 90,95, 96, 97, 98 or 99%. The antagonist may be resistant to protease, forexample one or more of the proteases as herein described, for exampleunder a set of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR1 sequence that is at least 50% identical tothe CDR1 sequence of DOM1h-131-206 (shown in FIG. 3) and a CDR2 sequencethat is at least 50% identical to the CDR2 sequence of DOM1h-131-206. Inone embodiment, the CDR sequence identity of one or both CDRs is atleast 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%respectively. The antagonist may be resistant to protease, for exampleone or more of the proteases as herein described, for example under aset of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR1 sequence that is at least 50% identical tothe CDR1 sequence of DOM1h-131-206 (shown in FIG. 3) and a CDR3 sequencethat is at least 50% identical to the CDR3 sequence of DOM1h-131-206. Inone embodiment, the CDR sequence identity of one or both CDRs is atleast 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%respectively. The antagonist may be resistant to protease, for exampleone or more of the proteases as herein described, for example under aset of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR2 sequence that is at least 50% identical tothe CDR2 sequence of DOM1h-131-206 (shown in FIG. 3) and a CDR3 sequencethat is at least 50% identical to the CDR3 sequence of DOM1h-131-206. Inone embodiment, the CDR sequence identity of one or both CDRs is atleast 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%respectively. The antagonist may be resistant to protease, for exampleone or more of the proteases as herein described, for example under aset of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist having a CDR1 sequence that is at least 50% identical tothe CDR1 sequence of DOM1h-131-206 (shown in FIG. 3) and a CDR2 sequencethat is at least 50% identical to the CDR2 sequence of DOM1h-131-206 anda CDR3 sequence that is at least 50% identical to the CDR3 sequence ofDOM1h-131-206. In one embodiment, the CDR sequence identity of one ortwo or each of the CDRs is at least 55, 60, 65, 70, 75, 80, 85, 90, 95,96, 97, 98 or 99% respectively. The antagonist may be resistant toprotease, for example one or more of the proteases as herein described,for example under a set of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist comprising an immunoglobulin single variable domaincomprising the sequence of CDR1, CDR2, and/or CDR3 (e.g., CDR1, CDR2,CDR3, CDR1 and 2, CDR1 and 3, CDR2 and 3 or CDR1, 2 and 3) ofDOM1h-131-206 (shown in FIG. 3). The antagonist may be resistant toprotease, for example one or more of the proteases as herein described,for example under a set of conditions as herein described.

In one aspect, the invention provides a TNFα receptor type 1 (TNFR1;p55) antagonist that competes with DOM1h-131-511-206 for binding toTNFR1. Thus, the antagonist may bind the same epitope as DOM1h-131-206or an overlapping epitope. In one embodiment, the antagonist comprisesan immunoglobulin single variable domain having an amino acid sequencethat is at least 93% identical to the amino acid sequence ofDOM1h-131-206 (shown in FIG. 3). In one embodiment, the percent identityis at least 94, 95, 96, 97, 98 or 99%. The antagonist may be resistantto protease, for example one or more of the proteases as hereindescribed, for example under a set of conditions as herein described. Inone embodiment, the antagonist is an antibody or antigen-bindingfragment thereof, such as a monovalent antigen-binding fragment (e.g.,scFv, Fab, Fab′, DAB™) that has binding specificity for TNFR1. Otherexamples of antagonists are ligands described herein that bind TNFR1.The ligands may comprise an immunoglobulin single variable domain ordomain antibody (DAB™) that has binding specificity for TNFR1, or thecomplementarity determining regions of such a DAB™ in a suitable format.In some embodiments, the ligand is a DAB™ monomer that consistsessentially of, or consists of, an immunoglobulin single variable domainor DAB™ that has binding specificity for TNFR1. In other embodiments,the ligand is a polypeptide that comprises a DAB™ (or the CDRs of aDAB™) in a suitable format, such as an antibody format.

Some antagonist of TNFR1 of the invention do not inhibit binding of TNFαto TNFR1, but do inhibit signal transduction mediated through TNFR1. Forexample, an antagonist of TNFR1 can inhibit TNFα-induced clustering ofTNFR1, which precedes signal transduction through TNFR1. Suchantagonists provide several advantages. For example, in the presence ofsuch an antagonist, TNFα can bind TNFR1 expressed on the surface ofcells and be removed from the cellular environment, but TNFR1 mediatedsignal transduction will not be activated. Thus, TNFR1 signal-inducedproduction of additional TNFα and other mediators of inflammation willbe inhibited. Similarly, antagonists of TNFR1 that bind TNFR1 andinhibit signal transduction mediated through TNFR1, but do no inhibitbinding of TNFα to TNFR1, will not inhibit the TNFα-binding andinhibiting activity of endogenously produced soluble TNFR1. Accordingly,administering such an antagonist to a mammal in need thereof cancomplement the endogenous regulatory pathways that inhibit the activityTNFα and the activity of TNFR1 in vivo. The invention also relates toligands that (i) bind TNFR1 (e.g., in Domain1), (ii) antagonize theactivation of TNFR1 mediated signal transduction, and (iii) do notinhibit the binding of TNFα to TNFR1. Such a ligand binds soluble TNFR1and does not prevent the soluble receptor from binding TNFα, and thusadministering such an antagonist to a mammal in need thereof cancomplement the endogenous regulatory pathways that inhibit the activityTNFα in vivo by increasing the half-life of the soluble receptor in theserum. These advantages are particularly relevant to ligands that havebeen formatted to have a larger hydrodynamic size, for example, byattachment of a PEG group, serum albumin, transferrin, transferrinreceptor or at least the transferrin-binding portion thereof, anantibody Fc region, or by conjugation to an antibody domain. Forexample, an agent (e.g., polypeptide, variable domain or antagonist)that i) binds TNFR1 (e.g., in Domain1), (ii) antagonizes the activationof TNFR1 mediated signal transduction, and (iii) does not inhibit thebinding of TNFα to TNFR1, such as a DAB™ monomer, can be formatted as alarger antigen-binding fragment of an antibody or as and antibody (e.g.,formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv). The hydrodynamicsize of a ligand and its serum half-life can also be increased byconjugating or linking a TNFR1 binding agent (antagonist; variabledomain) to a binding domain (e.g., antibody or antibody fragment) thatbinds an antigen or epitope that increases half-live in vivo, asdescribed herein (see, Annex 1 of WO2006038027 incorporated herein byreference in its entirety). For example, the TNFR1 binding agent (e.g.,polypeptide) can be conjugated or linked to an anti-serum albumin oranti-neonatal Fc receptor antibody or antibody fragment, e.g. an anti-SAor anti-neonatal Fc receptor DAB™, Fab, Fab′ or scFv, or to an anti-SAaffibody or anti-neonatal Fc receptor affibody.

Examples of suitable albumin, albumin fragments or albumin variants foruse in a TNFR1-binding ligand according to the invention are describedin WO 2005/077042A2 and WO2006038027, which are incorporated herein byreference in their entirety.

In other embodiments of the invention described throughout thisdisclosure, instead of the use of a “DAB™” in an antagonist or ligand ofthe invention, it is contemplated that the skilled addressee can use adomain that comprises the CDRs of a DAB™ that binds TNFR1 (e.g., CDRsgrafted onto a suitable protein scaffold or skeleton, e.g. an affibody,an SpA scaffold, an LDL receptor class A domain or an EGF domain) or canbe a protein domain comprising a binding site for TNFR1, e.g., whereinthe domain is selected from an affibody, an SpA domain, an LDL receptorclass A domain or an EGF domain. The disclosure as a whole is to beconstrued accordingly to provide disclosure of antagonists, ligands andmethods using such domains in place of a DAB™.

Polypeptides, immunoglobulin single variable domains and antagonists ofthe invention may be resistant to one or more of the following: serineprotease, cysteine protease, aspartate proteases, thiol proteases,matrix metalloprotease, carboxypeptidase (e.g., carboxypeptidase A,carboxypeptidase B), trypsin, chymotrypsin, pepsin, papain, elastase,leukozyme, pancreatin, thrombin, plasmin, cathepsins (e.g., cathepsinG), proteinase (e.g., proteinase 1, proteinase 2, proteinase 3),thermolysin, chymosin, enteropeptidase, caspase (e.g., caspase 1,caspase 2, caspase 4, caspase 5, caspase 9, caspase 12, caspase 13),calpain, ficain, clostripain, actinidain, bromelain, and separase. Inparticular embodiments, the protease is trypsin, elastase or leucozyme.The protease can also be provided by a biological extract, biologicalhomogenate or biological preparation. In one embodiment, the protease isa protease found in sputum, mucus (e.g., gastric mucus, nasal mucus,bronchial mucus), bronchoalveolar lavage, lung homogenate, lung extract,pancreatic extract, gastric fluid, saliva or tears. In one embodiment,the protease is one found in the eye and/or tears. In one embodiment,the protease is a non-bacterial protease. In an embodiment, the proteaseis an animal, e.g., mammalian, e.g., human, protease. In an embodiment,the protease is a GI tract protease or a pulmonary tissue protease,e.g., a GI tract protease or a pulmonary tissue protease found inhumans. Such protease listed here can also be used in the methodsdescribed herein involving exposure of a repertoire of library to aprotease.

In one aspect, the invention provides a protease resistantimmunoglobulin single variable domain comprising a TNFα receptor type 1(TNFR1; p55) binding site, wherein the variable domain is resistant toprotease, e.g. trypsin, when incubated with (i) a concentration (c) ofat least 10 micrograms/ml protease at 37° C. for time (t) of at leastone hour; or (ii) a concentration (c′) of at least 40 micrograms/mlprotease at 30° C. for time (t) of at least one hour, wherein thevariable domain comprises and amino acid sequence that is at least 90%identical to the amino acid sequence of DOM1h-131-206 (shown in FIG. 3).In one embodiment, the ratio (on a mole/mole basis) of protease, e.g.trypsin, to variable domain is 8,000 to 80,000 protease:variable domain,e.g. when C is 10 micrograms/ml, the ratio is 800 to 80,000protease:variable domain; or when C or C′ is 100 micrograms/ml, theratio is 8,000 to 80,000 protease:variable domain. In one embodiment theratio (on a weight/weight, e.g. microgram/microgram basis) of protease(e.g., trypsin) to variable domain is 16,000 to 160,000protease:variable domain e.g. when C is 10 micrograms/ml, the ratio is1,600 to 160,000 protease:variable domain; or when C or C′ is 100micrograms/ml, the ratio is 1,6000 to 160,000 protease:variable domain.In one embodiment, the concentration (c or c′) is at least 100 or 1000micrograms/ml protease. In one embodiment, the concentration (c or c′)is at least 100 or 1000 micrograms/ml protease. Reference is made to thedescription herein of the conditions suitable for proteolytic activityof the protease for use when working with repertoires or libraries ofpeptides or polypeptides (e.g., w/w parameters). These conditions can beused for conditions to determine the protease resistance of a particularimmunoglobulin single variable domain. In one embodiment, time (t) is oris about one, three or 24 hours or overnight (e.g., about 12-16 hours).In one embodiment, the variable domain is resistant under conditions (i)and the concentration (c) is or is about 10 or 100 micrograms/mlprotease and time (t) is 1 hour. In one embodiment, the variable domainis resistant under conditions (ii) and the concentration (c′) is or isabout 40 micrograms/ml protease and time (t) is or is about 3 hours. Inone embodiment, the protease is selected from trypsin, elastase,leucozyme and pancreatin. In one embodiment, the protease is trypsin. Inone embodiment, the protease is a protease found in sputum, mucus (e.g.,gastric mucus, nasal mucus, bronchial mucus), bronchoalveolar lavage,lung homogenate, lung extract, pancreatic extract, gastric fluid, salivaor tears. In one embodiment, the protease is one found in the eye and/ortears. In one embodiment, the protease is a non-bacterial protease. Inan embodiment, the protease is an animal, e.g., mammalian, e.g., human,protease. In an embodiment, the protease is a GI tract protease or apulmonary tissue protease, e.g., a GI tract protease or a pulmonarytissue protease found in humans. Such protease listed here can also beused in the methods described herein involving exposure of a repertoireof library to a protease.

In one embodiment, the variable domain is resistant to trypsin and/or atleast one other protease selected from elastase, leucozyme andpancreatin. For example, resistance is to trypsin and elastase; trypsinand leucozyme; trypsin and pacreatin; trypsin, elastase and leucozyme;trypsin, elastase and pancreatin; trypsin, elastase, pancreatin andleucozyme; or trypsin, pancreatin and leucozyme.

In one embodiment, the variable domain is displayed on bacteriophagewhen incubated under condition (i) or (ii), for example at a phagelibrary size of 10⁶ to 10¹³, e.g. 10⁸ to 10¹² replicative units(infective virions).

In one embodiment, the variable domain specifically binds TNFR1following incubation under condition (i) or (ii), e.g. assessed usingBIACORE™ or ELISA, e.g. phage ELISA or monoclonal phage ELISA.

In one embodiment, the variable domains of the invention specificallybind protein A or protein L. In one embodiment, specific binding toprotein A or L is present following incubation under condition (i) or(ii).

In one embodiment, the variable domains of the invention may have anOD₄₅₀ reading in ELISA, e.g. phage ELISA or monoclonal phage ELISA) ofat least 0.404, e.g., following incubation under condition (i) or (ii).

In one embodiment, the variable domains of the invention display(substantially) a single band in gel electrophoresis, e.g. followingincubation under condition (i) or (ii).

In certain embodiments, the invention provides a TNFR1 antagonist thatis a dual-specific ligand that comprises a first DAB™ according to theinvention that binds TNFR1 and a second DAB™ that has the same or adifferent binding specificity from the first DAB™. The second DAB™ maybind a target selected from ApoE, Apo-SAA, BDNF, Cardiotrophin-1, CEA,CD40, CD40 Ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78,Eotaxin, Eotaxin-2, Exodus-2, FAPα, FGF-acidic, FGF-basic, fibroblastgrowth factor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF,GF-β1, human serum albumin, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β,IL-1 receptor, IL-1 receptor type 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocytegrowth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerianinhibitory substance, monocyte colony inhibitory factor, monocyteattractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF),MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP-1β,MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M,PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stemcell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosisfactor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1,TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGFreceptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309,HER 1, HER 2, HER 3, HER 4, serum albumin, vWF, amyloid proteins (e.g.,amyloid alpha), MMP12, PDK1, IgE, IL-13Rα1, IL-13Ra2, IL-15, IL-15R,IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2, CD4,CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD40L, CD56, CD138, ALK5,EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1), chymase,FGF, Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2,Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFNa, 1-309, integrins,L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil elastase,osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1, siglec8, TARC, TGFb,Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF, VLA-4, VCAM, α4β7, CCR2,CCR3, CCR4, CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF,amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.

In one example, the dual-specific ligand comprises a first DAB™ thatbinds a first epitope on TNFR1 and a second DAB™ that binds an epitopeon a different target. In another example, the second DAB™ binds anepitope on serum albumin.

In other embodiments, the ligand is a multispecific ligand thatcomprises a first epitope binding domain that has binding specificityfor TNFR1 and at least one other epitope binding domain that has bindingspecificity different from the first epitope binding domain. Forexample, the first epitope binding domain can be a DAB™ that binds TNFR1or can be a domain that comprises the CDRs of a DAB™ that binds TNFR1(e.g., CDRs grafted onto a suitable protein scaffold or skeleton, e.g.,an affibody, an SpA scaffold, an LDL receptor class A domain or an EGFdomain) or can be a domain that binds TNFR1, wherein the domain isselected from an affibody, an SpA domain, an LDL receptor class A domainor an EGF domain).

In certain embodiments, the polypeptide, antagonist, ligand oranti-TNFR1 DAB™ monomer is characterized by one or more of thefollowing: 1) dissociates from human TNFR1 with a dissociation constant(K_(d)) of 50 nM to 20 pM, and a K_(off) rate constant of 5×10⁻¹ to1×10⁻⁷ s⁻¹ as determined by surface plasmon resonance; 2) inhibitsbinding of Tumor Necrosis Factor Alpha (TNFα) to TNFR1 with an IC50 of500 nM to 50 pM; 3) neutralizes human TNFR1 in a standard L929 cellassay with an ND50 of 500 nM to 50 pM; 4) antagonizes the activity ofthe TNFR1 in a standard cell assay with an ND₅₀ of ≦100 nM, and at aconcentration of ≦10 μM the DAB™ agonizes the activity of the TNFR1 by≦5% in the assay; 5) inhibits lethality in the mouseLPS/D-galactosamine-induced septic shock model; 6) resists aggregation;7) is secreted in a quantity of at least about 0.5 mg/L when expressedin E. coli or Pichia species (e.g., P. pastoris); 8) unfolds reversibly;9) has efficacy in a model of chronic inflammatory disease selected fromthe group consisting of mouse collagen-induced arthritis model, mouseΔARE model of arthritis, mouse ΔARE model of inflammatory bowel disease,mouse dextran sulfate sodium-induced model of inflammatory boweldisease, mouse tobacco smoke model of chronic obstructive pulmonarydisease, and suitable primate models (e.g., primate collagen-inducedarthritis model); and/or 10) has efficacy in treating, suppressing orpreventing a chronic inflammatory disease. Reference is made toWO2006038027 for details of assays and tests and parameters applicableto conditions (1) to (10), and this incorporated herein by reference.

In particular embodiments, the polypeptide, antagonist, ligand or DAB™monomer dissociates from human TNFR1 with a dissociation constant(K_(d)) of 50 nM to 20 pM, and a K_(off) rate constant of 5×10⁻¹ to1×10⁻⁷ s⁻¹ as determined by surface plasmon resonance; inhibits bindingof Tumor Necrosis Factor Alpha (TNFα) to TNFR1 with an IC50 of 500 nM to50 pM; and neutralizes human TNFR1 in a standard L929 cell assay with anND50 of 500 nM to 50 pM. In other particular embodiments, thepolypeptide, antagonist, ligand or DAB™ monomer dissociates from humanTNFR1 with a dissociation constant (K_(d)) of 50 nM to 20 pM, and aK_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ s⁻¹; inhibits binding of TumorNecrosis Factor Alpha (TNFα) to TNFR1 with an IC50 of 500 nM to 50 pM;and has efficacy in a model of chronic inflammatory disease selectedfrom the group consisting of mouse collagen-induced arthritis model,mouse ΔARE model of arthritis, mouse ΔARE model of inflammatory boweldisease, mouse dextran sulfate sodium-induced model of inflammatorybowel disease, mouse tobacco smoke model of chronic obstructivepulmonary disease, and suitable primate models (e.g., primatecollagen-induced arthritis model). In other particular embodiments, thepolypeptide, antagonist, ligand or DAB™ monomer dissociates from humanTNFR1 with a dissociation constant (K_(d)) of 50 nM to 20 pM, and aK_(off) rate constant of 5×10⁻¹ to 1×10⁻⁷ s⁻¹ as determined by surfaceplasmon resonance; neutralizes human TNFR1 in a standard L929 cell assaywith an ND50 of 500 nM to 50 pM; and antagonizes the activity of theTNFR1 in a standard cell assay with an ND₅₀ of ≦100 nM, and at aconcentration of ≦10 μM the DAB™ agonizes the activity of the TNFR1 by≦5% in the assay.

The protease resistant polypeptides, immunoglobulin single variabledomains and antagonists of the invention have utility in therapy,prophylaxis and diagnosis of disease or conditions in mammals, e.g.,humans. In particular, they have utility as the basis of drugs that arelikely to encounter proteases when administered to a patient, such as ahuman. For example, when administered to the GI tract (e.g., orally,sublingually, rectally administered), in which case the polypeptides,immunoglobulin single variable domains and antagonists may be subjectedto protease in one or more of the upper GI tract, lower GI tract, mouth,stomach, small intestine and large intestine. One embodiment, therefore,provides for a protease resistant polypeptide, immunoglobulin singlevariable domain or antagonist to be administered orally, sublingually orrectally to the GI tract of a patient to treat and/or prevent a diseaseor condition in the patient. For example, oral administration to apatient (e.g., a human patient) for the treatment and/or prevention of aTNF alpha-mediated condition or disease such as arthritis (e.g.,rheumatoid arthritis), IBD, psoriasis or Crohn's disease. In anotherexample, the polypeptide, variable domain or antagonist is likely toencounter protease when administered (e.g., by inhalation orintranasally) to pulmonary tissue (e.g., the lung or airways). Oneembodiment, therefore, provides for administration of the proteaseresistant polypeptide, immunoglobulin single variable domain orantagonist to a patient (e.g., to a human) by inhalation or intranasallyto pulmonary tissue of the patient to treat and/or prevent a disease orcondition in the patient. Such condition may be asthma (e.g., allergicasthma), COPD, influenza or any other pulmonary disease or conditiondisclosed in WO2006038027, incorporated herein by reference. In anotherexample, the polypeptide, variable domain or antagonist is likely toencounter protease when administered (e.g., by intraocular injection oras eye drops) to an eye of a patient. One embodiment, therefore,provides for ocular administration of the protease resistantpolypeptide, immunoglobulin single variable domain or antagonist to apatient (e.g., to a human) by to treat and/or prevent a disease orcondition (e.g., a disease or condition of the eye) in the patient.Administration could be topical administration to the eye, in the formof eye drops or by injection into the eye, e.g. into the vitreoushumour.

The antagonists, polypeptides and immunoglobulin single variable domainsaccording to the invention may display improved or relatively highmelting temperatures (Tm), providing enhanced stability. High affinitytarget binding may also or alternatively be a feature of theantagonists, polypeptides and variable domains. One or more of thesefeatures, combined with protease resistance, makes the antagonists,variable domains and polypeptides amenable to use as drugs in mammals,such as humans, where proteases are likely to be encountered, e.g. forGI tract or pulmonary tissue administration.

Thus, in one aspect, the invention provides the TNFR1 antagonist fororal delivery. In one aspect, the invention provides the TNFR1antagonist for delivery to the GI tract of a patient. In one aspect, theinvention provides the use of the TNFR1 antagonist in the manufacture ofa medicament for oral delivery. In one aspect, the invention providesthe use of the TNFR1 antagonist in the manufacture of a medicament fordelivery to the GI tract of a patient. In one embodiment, the variabledomain is resistant to trypsin and/or at least one other proteaseselected from elastase, leucozyme and pancreatin. For example,resistance is to trypsin and elastase; trypsin and leucozyme; trypsinand pacreatin; trypsin, elastase and leucozyme; trypsin, elastase andpancreatin; trypsin, elastase, pancreatin and leucozyme; or trypsin,pancreatin and leucozyme.

In one aspect, the invention provides the TNFR1 antagonist for pulmonarydelivery. In one aspect, the invention provides the TNFR1 antagonist fordelivery to the lung of a patient. In one aspect, the invention providesthe use of the TNFR1 antagonist in the manufacture of a medicament forpulmonary delivery. In one aspect, the invention provides the use of theTNFR1 antagonist in the manufacture of a medicament for delivery to thelung of a patient. In one embodiment, the variable domain is resistantto leucozyme.

In one aspect, the invention provides a method of oral delivery ordelivery of a medicament to the GI tract of a patient or to the lung orpulmonary tissue of a patient, wherein the method comprisesadministering to the patient a pharmaceutically effective amount of aTNFR1 antagonist of the invention.

In one aspect, the invention provides the TNFR1 antagonist of theinvention for treating and/or prophylaxis of an inflammatory condition.In one aspect, the invention provides the use of the TNFR1 antagonist inthe manufacture of a medicament for treating and/or prophylaxis of aninflammatory condition. In one embodiment, the condition is selectedfrom the group consisting of arthritis, multiple sclerosis, inflammatorybowel disease and chronic obstructive pulmonary disease. For example,said arthritis is rheumatoid arthritis or juvenile rheumatoid arthritis.For example, said inflammatory bowel disease is selected from the groupconsisting of Crohn's disease and ulcerative colitis. For example, saidchronic obstructive pulmonary disease is selected from the groupconsisting of chronic bronchitis, chronic obstructive bronchitis andemphysema. For example, said pneumonia is bacterial pneumonia. Forexample, said bacterial pneumonia is Staphylococcal pneumonia.

In one aspect, the invention provides the TNFR1 antagonist for treatingand/or prophylaxis of a respiratory disease. In one aspect, theinvention provides the use of the TNFR1 antagonist in the manufacture ofa medicament for treating and/or prophylaxis of a respiratory disease.For example, said respiratory disease is selected from the groupconsisting of lung inflammation, chronic obstructive pulmonary disease,asthma, pneumonia, hypersensitivity pneumonitis, pulmonary infiltratewith eosinophilia, environmental lung disease, pneumonia,bronchiectasis, cystic fibrosis, interstitial lung disease, primarypulmonary hypertension, pulmonary thromboembolism, disorders of thepleura, disorders of the mediastinum, disorders of the diaphragm,hypoventilation, hyperventilation, sleep apnea, acute respiratorydistress syndrome, mesothelioma, sarcoma, graft rejection, graft versushost disease, lung cancer, allergic rhinitis, allergy, asbestosis,aspergilloma, aspergillosis, bronchiectasis, chronic bronchitis,emphysema, eosinophilic pneumonia, idiopathic pulmonary fibrosis,invasive pneumococcal disease, influenza, nontuberculous mycobacteria,pleural effusion, pneumoconiosis, pneumocytosis, pneumonia, pulmonaryactinomycosis, pulmonary alveolar proteinosis, pulmonary anthrax,pulmonary edema, pulmonary embolus, pulmonary inflammation, pulmonaryhistiocytosis X, pulmonary hypertension, pulmonary nocardiosis,pulmonary tuberculosis, pulmonary veno-occlusive disease, rheumatoidlung disease, sarcoidosis, and Wegener's granulomatosis. For example,the disease is chronic obstructive pulmonary disease (COPD). Forexample, the disease is asthma.

An antagonist of the invention comprising an agent that inhibits TNFR1(e.g., wherein the agent is selected from the group consisting ofantibody fragments (e.g., Fab fragment, Fab′ fragment, Fv fragment(e.g., scFv, disulfide bonded Fv), F(ab′)₂ fragment, DAB™), ligands andDAB™ monomers and multimers (e.g., homo- or heterodimers) can be locallyadministered to pulmonary tissue (e.g., lung) of a subject using anysuitable method. For example, an agent can be locally administered topulmonary tissue via inhalation or intranasal administration. Forinhalation or intranasal administration, the antagonist of TNFR1 can beadministered using a nebulizer, inhaler, atomizer, aerosolizer, mister,dry powder inhaler, metered dose inhaler, metered dose sprayer, metereddose mister, metered dose atomizer, or other suitable inhaler orintranasal delivery device. Thus, in one embodiment, the inventionprovides a pulmonary delivery device containing the TNFR1 antagonist. Inone embodiment, the device is an inhaler or an intranasal deliverydevice.

In one aspect, the invention provides an oral formulation comprising theTNFR1 antagonist. The formulation can be a tablet, pill, capsule, liquidor syrup.

In one embodiment, the invention provides a pulmonary formulation fordelivery to the lung, wherein the formulation comprise an antagonist,polypeptide or variable domain of the invention with a particle sizerange of less than 5 microns, for example less than 4.5, 4, 3.5 or 3microns (e.g., when in Britton-Robinson buffer, e.g. at a pH of 6.5 to8.0, e.g. at a pH of 7 to 7.5, e.g. at pH7 or at pH7.5).

In one embodiment, the formulations and compositions of the inventionare provided at a pH from 6.5 to 8.0, for example 7 to 7.5, for example7, for example 7.5.

Variable domains according to any aspect of the invention may have a Tmof at least 50° C., or at least 55° C., or at least 60° C., or at least65° C., or at least 70° C. An antagonist, use, method, device orformulation of the invention may comprise such a variable domain.

In one aspect of the invention, the polypeptides, variable domains,antagonists, compositions or formulations of the invention aresubstantially stable after incubation (at a concentration of polypeptideor variable domain of 1 mg/ml) at 37 to 50° C. for 14 days inBritton-Robinson buffer. In one embodiment, at least 65, 70, 75, 80, 85,86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the polypeptide,antagonist or variable domain remains unaggregated after such incubationat 37 degrees C. In one embodiment, at least 65, 70, 75, 80, 85, 86, 87,88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of the polypeptide orvariable domain remains monomeric after such incubation at 37 degrees C.In one embodiment, at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98,99% of the polypeptide, antagonist or variable domain remainsunaggregated after such incubation at 50 degrees C. In one embodiment,at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% of thepolypeptide or variable domain remains monomeric after such incubationat 50 degrees C. In one embodiment, no aggregation of the polypeptides,variable domains, antagonists is seen after any one of such incubations.In one embodiment, the pI of the polypeptide or variable domain remainsunchanged or substantially unchanged after incubation at 37 degrees C.at a concentration of polypeptide or variable domain of 1 mg/ml inBritton-Robinson buffer.

In one aspect of the invention, the polypeptides, variable domains,antagonists, compositions or formulations of the invention aresubstantially stable after incubation (at a concentration of polypeptideor variable domain of 100 mg/ml) at 4° C. for 7 days in Britton-Robinsonbuffer at a pH of 7 to 7.5 (e.g., at pH7 or pH7.5). In one embodiment,at least 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or 99.5% of thepolypeptide, antagonist or variable domain remains unaggregated aftersuch incubation. In one embodiment, at least 95, 95.5, 96, 96.5, 97,97.5, 98, 98.5, 99 or 99.5% of the polypeptide or variable domainremains monomeric after such incubation. In one embodiment, noaggregation of the polypeptides, variable domains, antagonists is seenafter any one of such incubations.

In one aspect of the invention, the polypeptides, variable domains,antagonists, compositions or formulations of the invention aresubstantially stable after nebulisation (at a concentration ofpolypeptide or variable domain of 40 mg/ml) e.g., at room temperature,20 degrees C. or 37° C., for 1 hour, e.g. in a jet nebuliser, e.g. aPARI LC+™ cup. In one embodiment, at least 65, 70, 75, 80, 85, 86, 87,88, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or99.5% of the polypeptide, antagonist or variable domain remainsunaggregated after such nebulisation. In one embodiment, at least 65,70, 75, 80, 85, 86, 87, 88, 90, 91, 92, 93, 94, 95, 95.5, 96, 96.5, 97,97.5, 98, 98.5, 99 or 99.5% of the polypeptide or variable domainremains monomeric after such nebulisation. In one embodiment, noaggregation of the polypeptides, variable domains, antagonists is seenafter any one of such nebulisation.

Variable domains according to any aspect of the invention may neutralizeTNFα stimulated IL-8 release in an MRC-5 cell assay with an ND50 of 2 nMto 50 pM. An antagonist, use, method, device or formulation of theinvention may comprise such a variable domain.

In one aspect, the invention provides an isolated or recombinant nucleicacid encoding a polypeptide comprising an immunoglobulin single variabledomain according to any aspect of the invention or encoding apolypeptide, antagonist or variable domain according to any aspect ofthe invention. In one aspect, the invention provides a vector comprisingthe nucleic acid. In one aspect, the invention provides a host cellcomprising the nucleic acid or the vector. In one aspect, the inventionprovides a method of producing polypeptide comprising an immunoglobulinsingle variable domain, the method comprising maintaining the host cellunder conditions suitable for expression of said nucleic acid or vector,whereby a polypeptide comprising an immunoglobulin single variabledomain is produced. The method may further comprise isolating thepolypeptide, variable domain or antagonist and optionally producing avariant, e.g. a mutated variant, having an improved affinity and/or ND50than the isolated polypeptide, variable domain or antagonist. Techniquesfor improving binding affinity of immunoglobulin single variable domainare known in the art, e.g. techniques for affinity maturation.

In one aspect, the invention provides a pharmaceutical compositioncomprising an immunoglobulin single variable domain, polypeptide or anantagonist of any aspect of the invention, and a pharmaceuticallyacceptable carrier, excipient or diluent.

In one embodiment, the immunoglobulin single variable domain or theantagonist of any aspect of the invention comprises an antibody constantdomain, for example, an antibody Fc, optionally wherein the N-terminusof the Fc is linked (optionally directly linked) to the C-terminus ofthe variable domain.

The polypeptide or variable domain of the invention can be isolatedand/or recombinant.

There is herein described a method for selecting a protease resistantpeptide or polypeptide. The method comprises providing a repertoire ofpeptides or polypeptides, combining the repertoire and a protease underconditions suitable for protease activity, and recovering a peptide orpolypeptide that has a desired biological activity (e.g., specificbinding to TNFR1), whereby a protease resistant peptide or polypeptideis selected.

The repertoire and the protease are generally incubated for a period ofat least about 30 minutes. Any desired protease can be used in themethod, such as one or more of the following, serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actinidain, bromelain, and separase. In particular embodiments, theprotease is trypsin, elastase or leucozyme. The protease can also beprovided by a biological extract, biological homogenate or biologicalpreparation. If desired, the method further comprises adding a proteaseinhibitor to the combination of the repertoire and the protease afterincubation is complete.

In some embodiments, a peptide or polypeptide that has a desiredbiological activity is recovered base on a binding activity. Forexample, the peptide or polypeptide can be recovered based on binding ageneric ligand, such as protein A, protein G or protein L. The bindingactivity can also be specific binding to a target ligand. Exemplarytarget ligands include ApoE, Apo-SAA, BDNF, Cardiotrophin-1, CEA, CD40,CD40 Ligand, CD56, CD38, CD138, EGF, EGF receptor, ENA-78, Eotaxin,Eotaxin-2, Exodus-2, FAPα, FGF-acidic, FGF-basic, fibroblast growthfactor-10, FLT3 ligand, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-β1,human serum albumin, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-1receptor, IL-1 receptor type 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8(72 a.a.), IL-8 (77 a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15,IL-16, IL-17, IL-18 (IGIF), Inhibin α, Inhibin β, IP-10, keratinocytegrowth factor-2 (KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerianinhibitory substance, monocyte colony inhibitory factor, monocyteattractant protein, M-CSF, MDC (67 a.a.), MDC (69 a.a.), MCP-1 (MCAF),MCP-2, MCP-3, MCP-4, MDC (67 a.a.), MDC (69 a.a.), MIG, MIP-1α, MIP-1β,MIP-3α, MIP-3β, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),NAP-2, Neurturin, Nerve growth factor, β-NGF, NT-3, NT-4, Oncostatin M,PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stemcell factor (SCF), TARC, TGF-α, TGF-β, TGF-β2, TGF-β3, tumour necrosisfactor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1,TPO, VEGF, VEGF A, VEGF B, VEGF C, VEGF D, VEGF receptor 1, VEGFreceptor 2, VEGF receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1, 1-309,HER 1, HER 2, HER 3, HER 4, serum albumin, vWF, amyloid proteins (e.g.,amyloid alpha), MMP12, PDK1, IgE, IL-13Rα1, IL-13Ra2, IL-15, IL-15R,IL-16, IL-17R, IL-17, IL-18, IL-18R, IL-23 IL-23R, IL-25, CD2, CD4,CD11a, CD23, CD25, CD27, CD28, CD30, CD40, CD40L, CD56, CD138, ALK5,EGFR, FcER1, TGFb, CCL2, CCL18, CEA, CR8, CTGF, CXCL12 (SDF-1), chymase,FGF, Furin, Endothelin-1, Eotaxins (e.g., Eotaxin, Eotaxin-2,Eotaxin-3), GM-CSF, ICAM-1, ICOS, IgE, IFNa, 1-309, integrins,L-selectin, MIF, MIP4, MDC, MCP-1, MMPs, neutrophil elastase,osteopontin, OX-40, PARC, PD-1, RANTES, SCF, SDF-1, siglec8, TARC, TGFb,Thrombin, Tim-1, TNF, TRANCE, Tryptase, VEGF, VLA-4, VCAM, α4β7, CCR2,CCR3, CCR4, CCR5, CCR7, CCR8, alphavbeta6, alphavbeta8, cMET, CD8, vWF,amyloid proteins (e.g., amyloid alpha), MMP12, PDK1, and IgE.

In particular embodiments, the peptide or polypeptide is recovered bypanning.

In some embodiments, the repertoire comprises a display system. Forexample, the display system can be bacteriophage display, ribosomedisplay, emulsion compartmentalization and display, yeast display,puromycin display, bacterial display, display on plasmid, or covalentdisplay. Exemplary display systems link coding function of a nucleicacid and functional characteristics of the peptide or polypeptideencoded by the nucleic acid. In particular embodiments, the displaysystem comprises replicable genetic packages.

In some embodiments, the display system comprises bacteriophage display.For example, the bacteriophage can be fd, M13, lambda, MS2 or T7. Inparticular embodiments, the bacteriophage display system is multivalent.In some embodiments, the peptide or polypeptide is displayed as a pIIIfusion protein.

In other embodiments, the method further comprises amplifying thenucleic acid encoding a peptide or polypeptide that has a desiredbiological activity. In particular embodiments, the nucleic acid isamplified by phage amplification, cell growth or polymerase chainreaction.

In some embodiments, the repertoire is a repertoire of immunoglobulinsingle variable domains. In particular embodiments, the immunoglobulinsingle variable domain is a heavy chain variable domain. In moreparticular embodiments, the heavy chain variable domain is a human heavychain variable domain. In other embodiments, the immunoglobulin singlevariable domain is a light chain variable domain. In particularembodiments, the light chain variable domain is a human light chainvariable domain.

In another aspect, there is provided a method for selecting a peptide orpolypeptide that binds a target ligand (e.g., TNFR1) with high affinityfrom a repertoire of peptides or polypeptides. The method comprisesproviding a repertoire of peptides or polypeptides, combining therepertoire and a protease under conditions suitable for proteaseactivity, and recovering a peptide or polypeptide that binds the targetligand.

The repertoire and the protease are generally incubated for a period ofat least about 30 minutes. Any desired protease can be used in themethod, such as one or more of the following, serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actinidain, bromelain, and separase. In particular embodiments, theprotease is trypsin, elastase or leucozyme. The protease can also beprovided by a biological extract, biological homogenate or biologicalpreparation. If desired, the method further comprises adding a proteaseinhibitor to the combination of the repertoire and the protease afterincubation is complete.

The peptide or polypeptide can be recovered based on binding any desiredtarget ligand, such as the target ligands disclosed herein (e.g.,TNFR1). In particular embodiments, the peptide or polypeptide isrecovered by panning.

In some embodiments, the repertoire comprises a display system. Forexample, the display system can be bacteriophage display, ribosomedisplay, emulsion compartmentalization and display, yeast display,puromycin display, bacterial display, display on plasmid, or covalentdisplay. Exemplary display systems link coding function of a nucleicacid and functional characteristics of the peptide or polypeptideencoded by the nucleic acid. In particular embodiments, the displaysystem comprises replicable genetic packages.

In some embodiments, the display system comprises bacteriophage display.For example, the bacteriophage can be fd, M13, lambda, MS2 or T7. Inparticular embodiments, the bacteriophage display system is multivalent.In some embodiments, the peptide or polypeptide is displayed as a pIIIfusion protein.

In other embodiments, the method further comprises amplifying thenucleic acid encoding a peptide or polypeptide that has a desiredbiological activity. In particular embodiments, the nucleic acid isamplified by phage amplification, cell growth or polymerase chainreaction.

In some embodiments, the repertoire is a repertoire of immunoglobulinsingle variable domains. In particular embodiments, the immunoglobulinsingle variable domain is a heavy chain variable domain. In moreparticular embodiments, the heavy chain variable domain is a human heavychain variable domain. In other embodiments, the immunoglobulin singlevariable domain is a light chain variable domain. In particularembodiments, the light chain variable domain is a human light chainvariable domain.

In another aspect, there is herein described a method of producing arepertoire of protease resistant peptides or polypeptides. The methodcomprises providing a repertoire of peptides or polypeptides, combiningthe repertoire of peptides or polypeptides and a protease under suitableconditions for protease activity, and recovering a plurality of peptidesor polypeptides that have a desired biological activity, whereby arepertoire of protease resistant peptides or polypeptides is produced.

In some embodiments, the repertoire and the protease are incubated for aperiod of at least about 30 minutes. For example, the protease used inthe method can be one or more of the following, serine protease,cysteine protease, aspartate proteases, thiol proteases, matrixmetalloprotease, carboxypeptidase (e.g., carboxypeptidase A,carboxypeptidase B), trypsin, chymotrypsin, pepsin, papain, elastase,leukozyme, pancreatin, thrombin, plasmin, cathepsins (e.g., cathepsinG), proteinase (e.g., proteinase 1, proteinase 2, proteinase 3),thermolysin, chymosin, enteropeptidase, caspase (e.g., caspase 1,caspase 2, caspase 4, caspase 5, caspase 9, caspase 12, caspase 13),calpain, ficain, clostripain, actinidain, bromelain, and separase. Inparticular embodiments, the protease is trypsin, elastase or leucozyme.The protease can also be provided by a biological extract, biologicalhomogenate or biological preparation. If desired, the method furthercomprises adding a protease inhibitor to the combination of therepertoire and the protease after incubation is complete.

In some embodiments, a plurality of peptides or polypeptides that have adesired biological activity is recovered based on a binding activity.For example, a plurality of peptides or polypeptides can be recoveredbased on binding a generic ligand, such as protein A, protein G orprotein L. The binding activity can also be specific binding to a targetligand, such as a target ligand described herein. In particularembodiments, a plurality of peptides or polypeptides that has thedesired biological activity is recovered by panning.

In some embodiments, the repertoire comprises a display system. Forexample, the display system can be bacteriophage display, ribosomedisplay, emulsion compartmentalization and display, yeast display,puromycin display, bacterial display, display on plasmid, or covalentdisplay. In particular embodiments, the display system links codingfunction of a nucleic acid and functional characteristics of the peptideor polypeptide encoded by the nucleic acid. In particular embodiments,the display system comprises replicable genetic packages.

In some embodiments, the display system comprises bacteriophage display.For example, the bacteriophage can be fd, M13, lambda, MS2 or T7. Inparticular embodiments, the bacteriophage display system is multivalent.In some embodiments, the peptide or polypeptide is displayed as a pIIIfusion protein.

In other embodiments, the method further comprises amplifying thenucleic acids encoding a plurality of peptides or polypeptides that havea desired biological activity. In particular embodiments, the nucleicacids are amplified by phage amplification, cell growth or polymerasechain reaction.

In some embodiments, the repertoire is a repertoire of immunoglobulinsingle variable domains. In particular embodiments, the immunoglobulinsingle variable domain is a heavy chain variable domain. In moreparticular embodiments, the heavy chain variable domain is a human heavychain variable domain. In other embodiments, the immunoglobulin singlevariable domain is a light chain variable domain. In particularembodiments, the light chain variable domain is a human light chainvariable domain.

In another aspect, there is herein described a method for selecting aprotease resistant polypeptide comprising an immunoglobulin singlevariable domain (DAB™) that binds a target ligand (e.g., TNFR1) from arepertoire. In one embodiment, the method comprises providing a phagedisplay system comprising a repertoire of polypeptides that comprise animmunoglobulin single variable domain, combining the phage displaysystem and a protease selected from the group consisting of elastase,leucozyme and trypsin, under conditions suitable for protease activity,and recovering a phage that displays a polypeptide comprising animmunoglobulin single variable domain that binds the target ligand.

In some embodiments, the protease is used at 100 μg/ml, and the combinedphage display system and protease are incubated at about 37° C.overnight.

In some embodiments, the phage that displays a polypeptide comprising animmunoglobulin single variable domain that binds the target ligand isrecovered by binding to said target. In other embodiments, the phagethat displays a polypeptide comprising an immunoglobulin single variabledomain that binds the target ligand is recovered by panning.

There is also described an isolated protease resistant peptide orpolypeptide selectable or selected by the methods described herein. In aparticular embodiment, there is provided an isolated protease (e.g.,trypsin, elastase, leucozyme) resistant immunoglobulin single variabledomain (e.g., human antibody heavy chain variable domain, human antibodylight chain variable domain) selectable or selected by the methodsdescribed herein.

There is further described herein an isolated or recombinant nucleicacid that encodes a protease resistant peptide or polypeptide (e.g.,trypsin-, elastase-, or leucozyme-resistant immunoglobulin singlevariable domain) selectable or selected by the methods described herein,and to vectors (e.g., expression vectors) and host cells that comprisethe nucleic acids.

There is further described herein a method for making a proteaseresistant peptide or polypeptide (e.g., trypsin-, elastase-, orleucozyme-resistant immunoglobulin single variable domain) selectable orselected by the methods described herein, comprising maintaining a hostcell that contains a recombinant nucleic acid encoding the proteaseresistant peptide or polypeptide under conditions suitable forexpression, whereby a protease resistant peptide or polypeptide isproduced.

There is further described herein a protease resistant peptide orpolypeptide (e.g., trypsin-, elastase-, or leucozyme-resistantimmunoglobulin single variable domain) selectable or selected by themethods described herein for use in medicine (e.g., for therapy ordiagnosis). There is further described herein the use of a proteaseresistant peptide or polypeptide (e.g., trypsin-, elastase-, orleucozyme-resistant immunoglobulin single variable domain) selectable orselected by the methods described herein for the manufacture of amedicament for treating disease. There is further described herein amethod of treating a disease, comprising administering to a subject inneed thereof, an effective amount of a protease resistant peptide orpolypeptide (e.g., trypsin-, elastase-, or leucozyme-resistantimmunoglobulin single variable domain) selectable or selected by themethods described herein.

There is further described herein a diagnostic kit for determine whetherTNFR1 is present in a sample or how much TNFR1 is present in a sample,comprising a polypeptide, immunoglobulin variable domain (DAB™) orantagonist of the invention and instructions for use (e.g., to determinethe presence and/or quantity of TNFR1 in the sample). In someembodiments, the kit further comprises one or more ancillary reagents,such as a suitable buffer or suitable detecting reagent (e.g., adetectably labeled antibody or antigen-binding fragment thereof thatbinds the polypeptide or DAB™ of the invention or a moiety associated orconjugated thereto.

The invention also relates to a device comprising a solid surface onwhich a polypeptide, antagonist or DAB™ of the invention is immobilizedsuch that the immobilized polypeptide or DAB™ binds TNFR1. Any suitablesolid surfaces on which an antibody or antigen-binding fragment thereofcan be immobilized can be used, for example, glass, plastics,carbohydrates (e.g., agarose beads). If desired the support can containor be modified to contain desired functional groups to facilitateimmobilization. The device, and or support, can have any suitable shape,for example, a sheet, rod, strip, plate, slide, bead, pellet, disk, gel,tube, sphere, chip, plate or dish, and the like. In some embodiments,the device is a dipstick.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the multiple cloning site of pDOM13 (akapDOM33), as shown in SEQ ID NO: 2 (GAS leader is shown in SEQ ID NO: 1)which was used to prepare a phage display repertoire.

FIG. 2 shows several NOVEX™ 10-20% TRICENE™ gels run with samples fromdifferent time points of DAB™s that were incubated with trypsin at 40ug/ml at 30° C. Samples were taken immediately before the addition oftrypsin, and then at one hour, three hours and 24 hours after theaddition of trypsin. The proteins were stained with 1× SUREBLUE™. Thegels illustrate that both DOM15-10 and DOM15-26-501 were significantlydigested during the first three hours of incubation with trypsin.Digestion of DOM15-26, DOM4-130-54 and DOM1h-131-511 only becameapparent after 24 hours of incubation with trypsin.

FIG. 3 is an illustration of the amino acid sequences of DOM1h-131-511(SEQ ID NO: 3) and 24 selected variants including DOM1h-131-206 (SEQ IDNO: 3). The amino acids that differ from the parent sequence in selectedclones are highlighted (those that are identical are marked by dots).The loops corresponding to CDR1, CDR2 and CDR3 are outlined with boxes.

FIG. 4 is an illustration of the amino acid sequences of DOM4-130-54(SEQ ID NO: 5) and 27 selected variants. The amino acids that differfrom the parent sequence in selected clones are highlighted (those thatare identical are marked by dots). The loops corresponding to CDR1, CDR2and CDR3 are outlined with boxes.

FIG. 5 is an illustration of the amino acid sequence of DOM15-26-555(SEQ ID NO: 7) and 21 selected variants. The amino acids that differfrom the parent sequence in selected clones are highlighted (those thatare identical are marked by dots). The loops corresponding to CDR1, CDR2and CDR3 are outlined with boxes. SYGA (SEQ ID NO: 6) is also shown.

FIG. 6 is an illustration of the amino acid sequence of DOM15-10 (SEQ IDNO: 10) and 16 selected variants. The amino acids that differ from theparent sequence in selected clones are highlighted (those that areidentical are marked by dots). The loops corresponding to CDR1, CDR2 andCDR3 are outlined with boxes. SYST (SEQ ID NO: 9) is also shown.

FIGS. 7A-7D are BIACORE™ traces showing bind of a parent DAB™,DOM1h-131-511 (FIG. 7A) and three variant DAB™s, DOM1h-131-203 (FIG.7B), DOM1h-131-204 (FIG. 7C) and DOM1h-131-206 (FIG. 7D), to immobilizedTNFR1 after incubation with different concentrations of trypsin (rangingfrom 0 to 100 μg/ml) overnight at 37° C. The results show that all threevariants are more resistant than the parent to proteolysis at highconcentrations of trypsin (100 ug/ml).

FIGS. 8A-8C are BIACORE™ traces showing binding of DAB™s DOM1h-131-511(FIG. 8A), DOM1h-131-202 (FIG. 8B) and DOM1h-131-206 (FIG. 8C) toimmobilized TNFR1 after incubation with elastase and leucozymeovernight. The DAB™s showed increased resistance to proteolysis comparedto the parent against both elastase and leucozyme.

FIG. 9 shows two 4-12% NOVEX™ Bis-Tris gels run with samples of DAB™sDOM1h-131-511, DOM1h-131-203, DOM1h-131-204, DOM1h-131-206,DOM1h-131-54, DOM1h-131-201, and DOM1h-131-202 before incubation withtrypsin and samples after incubation with 100 μg/ml of trypsin for 1hour, 3 hours and 24 hours.

FIGS. 10A-10C are BIACORE™ traces showing binding of DOM4-130-54 (FIG.10A), DOM4-130-201 (FIG. 10B) and DOM4-130-202 (FIG. 10C) to immobilizedIL-1R1 fusion protein after incubation with different concentrations oftrypsin (ranging from 0 to 100 μg/ml) overnight at 37° C. The resultsshow that both variants are more resistant than their parent toproteolysis at high concentrations of trypsin (100 μg/ml).

FIGS. 11A-11C are BIACORE™ traces showing binding of DOM4-130-54 (FIG.11A), DOM4-130-201 (FIG. 11B) and DOM4-130-202 (FIG. 11C) to immobilizedIL-1R1 fusion protein after incubation with elastase and leucozymeovernight. The DAB™s showed increased resistance to proteolysis comparedto parent against both proteases tested.

FIG. 12 is an illustration of the amino acid sequence of DOM15-26-555SEQ ID NO: 12 and 6 variants. The amino acids that differ from theparent sequence in selected clones are highlighted (those that areidentical are marked by dots). SYGA (SEQ ID NO: 11) is also shown.

FIGS. 13A and 13B are BIACORE™ traces showing binding of the parentDAB™, DOM15-26-555 (FIG. 13A) and the most protease resistant variant,DOM15-26-593 (FIG. 13B) to immobilized VEGF. The parent and the variantwere compared on the BIACORE™ for hVEGF binding at the DAB™concentration of 100 nM after incubation with trypsin at a concentrationof 200 μg/ml. The reaction was carried out for three hours or 24 hoursat 37° C. The results show that the variant is more resistant than theparent to proteolysis after 24 hours of trypsin treatment.

FIG. 14 is a graph showing effects of trypsin treatment on hVEGF bindingby DOM15-26-555 variants. The results clearly show that all variants aremore resistant than the parent (DOM15-26-555) to proteolysis after 24hours of trypsin treatment.

FIG. 15 shows two NOVEX™ 10-20% Tricine gels that were loaded with 15 μgof treated and untreated samples of DOM15-26-555 or DOM15-26-593.Samples were taken immediately before the addition of trypsin, and thenat one hour, three hours and 24 hours after the addition of trypsin. Theproteins were stained with 1× SUREBLUE™. The gels illustrate that thetrypsin resistance profile of DOM15-26-593 varied from the profile shownby the BIACORE™ experiment.

FIG. 16 is an illustration of the amino acid sequence of DOM15-10 (SEQID NO: 14) and a variant, DOM15-10-11. The amino acids that differ fromthe parent sequence in the variant are highlighted (those that areidentical are marked by dots). SYST (SEQ ID NO: 13) is also shown.

FIGS. 17A and 17B are BIACORE™ traces showing binding of the parent,DOM15-10 (FIG. 17A) and the variant, DOM15-10-11 (FIG. 17B), toimmobilized VEGF. The parent and the variant were compared on theBIACORE™ for hVEGF binding at the DAB™ concentration of 100 nM afterincubation with trypsin at a concentration of 200 μg/ml. The reactionwas carried out for one hour, three hours and 24 hours at 37° C. Theresults show that the variant is more resistant than the parent toproteolysis after 24 hours of trypsin treatment.

FIG. 18 shows two NOVEX™ 10-20% TRICENE™ gels that were loaded with 15μg of samples of DOM15-10 and DOM15-10-11. Samples were takenimmediately before the addition of trypsin, and then at one hour, threehours, and 24 hours after the addition of trypsin. The proteins werestained with SUREBLUE™ (1×). The results show that the binding activityseen in the BIACORE™ study directly reflects the protein's integrity.

FIGS. 19A-19L illustrate the nucleotide sequences of several nucleicacids (as shown in SEQ ID NO:s 15-67) encoding DAB™s that are variantsof DOM1h-131-511 or DOM4-130-54. The nucleotide sequences encode theamino acid sequences presented in FIG. 3 and FIG. 4, respectively.

FIGS. 20A-20E illustrate the nucleotide sequences of several nucleicacids (as shown in SEQ ID NO:s 68-102) encoding DAB™s that are variantsof DOM15-26-555 or DOM15-10. The nucleotide sequences encode the aminoacid sequences presented in FIG. 5 and FIG. 6, respectively.

FIG. 21 shows a vector map of pDOM 38.

FIG. 22: Shows a gel run on LABCHIP™ of DOM10-53-474 and DOM15-26-593proteins treated with trypsin at 25:1 DAB™:trypsin ratio at 30° C. fordifferent time points. Arrows show full length protein.

FIG. 23: Is a size exclusion chromatography trace showing the high levelof purity obtained for each sample after purification by MMCchromatography followed by anion exchange. The UV was monitored at 225nm and the column was run in 1×PBS with 10% ethanol (v/v). Thepercentage monomer was calculated by integration of the peak area withbaseline correction.

FIG. 24: Shows protease stability data for DOM1h-131-511, DOM1h-131-202and DOM1h-131-206.

FIG. 25: Is an SEC which illustrates 14 day stability data ofDOM1h-131-202, DOM1h-131-206 and DOM1h-131-511 in Britton-Robinsonbuffer at 37 and 50° C. The protein concentration for all the DAB™s was1 mg/ml. SEC was used to determine if any changes had occurred in theprotein during thermal stress and the amount of monomer left in solutionrelative to the time=0 (T0) sample.

FIGS. 26 A to I: Show SEC traces showing the effect of thermal stress(37 and 50° C.) on DOM1h-131-511 (A to C), -202 (D to F) and -206 (G toI). Also shown is the percentage of monomer left in solution relative tothe T=0 at the given time point.

FIG. 27: Shows IEF analysis of DOM1h-131-202, DOM1h-131-206 andDOM1h-131-511 at 24 hr, 48 hr and 7 and 14 days thermal stress. Thesamples had been incubated at either 37 or 50° C. in Britton-Robinsonbuffer.

FIG. 28: TNFR-1 RBA showing the effect of 14 days incubation ofDOM1h-131-202, DOM1h-131-206 and DOM1h-131-511 at 50° C. The proteinconcentration was assumed to be 1 mg/ml. A negative control DAB™ (VHdummy) which does not bind antigen is also shown.

FIG. 29: Illustrates Effects of storing A: DOM1h-131-202, B:DOM1h-131-206 and C: DOM1h-131-511 at ˜100 mg/ml for 7 days inBritton-Robinson buffer at +4° C. The UV was monitored at 280 nm.

FIG. 30: Shows data from Nebuliser testing of DOM1h-131-202,DOM1h-131-206 and DOM1h-131-511 in the PARI EFLOW™ and PARI LC+™. Theprotein concentration was 5 mg/ml in either Britton-Robinson buffer.

FIG. 31: Illustrates the Relative percentage changes in monomerconcentrations during nebulisation of DOM1h-131-202, DOM1h-131-206 andDOM1h-131-511 in Britton-Robinson buffer at 5 mg/ml.

FIG. 32: Shows SEC traces of DOM1h-131-206 and DOM1h-131-511 inBritton-Robinson buffer post nebulisation from the PARI LC+™.

FIG. 33: Shows SEC traces of DOM1h-131-206 during the nebulisationprocess over 1 hour at 40 mg/ml in PBS. The protein in both thenebuliser cup and aerosol are highly resistance to the effects of shearand thermal stress that may be experienced by the DAB™ duringnebulisation.

FIG. 34: Shows the sedimentation velocity curves for each of the threelead proteins (DOM1h-131-206 and DOM1h-131-511 and DOM1h-131-202). Thebimodal peak observed for the lower concentration sample ofDOM1h-131-206 is an artefact owing to a sample leak from the cell inthis instance.

FIG. 35: Shows the effect of buffer and device on nebulised droplet sizeof GSK 1995056A (DOM1h-131-511).

FIG. 36: Stability of GSK1995056A (DOM1h-131-511) after nebulisation invarious devices assessed by dimer formation as measured by SEC.

FIG. 37: Shows nebuliser testing of GSK1922567A (202), GSK1995057A (206)and GSK1995056A (511) in the PARI EFLOW™ and LC+. A) testing inBritton-Robinson buffer, B) testing in PEG1000/sucrose buffer.

FIG. 38: Depicts a TNF-α dose curve in the human TNFR1 receptor bindingassay. Each sample was tested as four replicates.

FIG. 39: Shows Inhibition by GSK1922567A (DOM1h-131-202), GSK1995057A(DOM1h-131-206) and GSK1995056A (DOM1h-131-511) in the human TNFRIreceptor binding assay. Each sample was tested as four replicates.

FIG. 40: Illustrates potency of the DOM15-26 and DOM15-26-593 DAB™s inthe VEGF RBA.

FIG. 41: Shows pharmacokinetics of DMS1529 (DOM 15-26-593) and DMS1545(DOM15-26-501) after single bolus dose i.v. administration to rats at 5mg/mg

FIG. 42A: Shows SEC-MALLs (Size exclusion chromatograph-multi-anglelaser light scattering) analysis of DMS1529 Fc fusion (DOM 15-26-593 Fcfusion) confirming monomeric properties. Two different batches are shownthat demonstrate similar properties with regard to refractive index(i.e. concentration; broken lines) and light scattering (solid lines).The line marked with the arrow signifies the molecular mass calculation.

FIG. 42B: Shows AUC (analytical ultracentrifugation) analysis of DMS1529Fc fusion (DOM 15-26-593 Fc fusion) confirming monomeric properties. Onebatch of material was tested at three different concentrations,approximating to 0.2, 0.5 & 1.0 mg/ml in PBS buffer. The analysis of thesedimentation rate confirmed a molecular mass of approx. 80 kDa.

FIG. 43: Shows DSC traces of DMS1529 (DOM15-26-593) and DOM15-26-501.

FIG. 44: Is a VEGF Binding ELISA for DMS1529 (DOM 15-26-593) before andafter, 10 freeze-thaw cycles on two different batches of material.

FIG. 45: Shows the consistency of DOM 15-26-593 SEC profile before andafter 10 freeze thaw cycles.

FIG. 46: Illustrates results from an accelerated stability study of theDMS 1529 fusion (DOM 15-26-593 Fc fusion); binding ELISA demonstratingactivity after 7 days incubation at the temperature shown.

FIG. 47A: Shows stability of DMS1529 (DOM 15-26-593) in cynomolgus serumafter 14 & 15 days incubation at 37° C.

FIG. 47B: Shows stability of DMS1529 (DOM 15-26-593) in human serumafter 14 & 15 days incubation at 37° C.

FIG. 48: Shows potency of DOM15-26 & DOM15-26-593 DAB™s as Fc fusions(DMS1564 & 1529 respectively) in the VEGF RBA.

FIG. 49: Illustrates inhibition of HUVEC cell proliferation by the DMS1529 fusion (DOM15-26-593 FC fusion).

FIG. 50: pDom33 vector map.

FIGS. 51A and B: Depict sequences (amino acid and nucleotide as shown inSEQ ID NO:s 103-221 and 263) of DAB™s that bind serum albumin.

FIGS. 52A-52C: Depict the amino acid sequence of the DOM15-26-593-Fcfusion (as shown in SEQ ID NO: 264), the amino acid sequence of thehuman IgG1 Fc portion (as shown in SEQ ID NO: 265) of theDOM15-26-593-Fc fusion and a nucleic acid sequence encoding theDOM15-26-593-Fc fusion (as shown in SEQ ID NO: 266).

DETAILED DESCRIPTION OF THE INVENTION

Within this specification the invention has been described, withreference to embodiments, in a way which enables a clear and concisespecification to be written. It is intended and should be appreciatedthat embodiments may be variously combined or separated without partingfrom the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art (e.g., in cell culture, molecular genetics, nucleic acidchemistry, hybridization techniques and biochemistry). Standardtechniques are used for molecular, genetic and biochemical methods (seegenerally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ded. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.and Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)Ed, John Wiley & Sons, Inc. which are incorporated herein by reference)and chemical methods.

As used herein, the term “antagonist of Tumor Necrosis Factor Receptor 1(TNFR1)” or “anti-TNFR1 antagonist” or the like refers to an agent(e.g., a molecule, a compound) which binds TNFR1 and can inhibit a(i.e., one or more) function of TNFR1. For example, an antagonist ofTNFR1 can inhibit the binding of TNFα to TNFR1 and/or inhibit signaltransduction mediated through TNFR1. Accordingly, TNFR1-mediatedprocesses and cellular responses (e.g., TNFα-induced cell death in astandard L929 cytotoxicity assay) can be inhibited with an antagonist ofTNFR1.

As used herein, “peptide” refers to about two to about 50 amino acidsthat are joined together via peptide bonds.

As used herein, “polypeptide” refers to at least about 50 amino acidsthat are joined together by peptide bonds. Polypeptides generallycomprise tertiary structure and fold into functional domains.

As used herein, a peptide or polypeptide (e.g. a domain antibody (DAB™))that is “resistant to protease degradation” is not substantiallydegraded by a protease when incubated with the protease under conditionssuitable for protease activity. A polypeptide (e.g., a DAB™) is notsubstantially degraded when no more than about 25%, no more than about20%, no more than about 15%, no more than about 14%, no more than about13%, no more than about 12%, no more than about 11%, no more than about10%, no more than about 9%, no more than about 8%, no more than about7%, no more than about 6%, no more than about 5%, no more than about 4%,no more than about 3%, no more that about 2%, no more than about 1%, orsubstantially none of the protein is degraded by protease afterincubation with the protease for about one hour at a temperaturesuitable for protease activity. For example at 37 or 50 degrees C.Protein degradation can be assessed using any suitable method, forexample, by SDS-PAGE or by functional assay (e.g., ligand binding) asdescribed herein.

As used herein, “display system” refers to a system in which acollection of polypeptides or peptides are accessible for selectionbased upon a desired characteristic, such as a physical, chemical orfunctional characteristic. The display system can be a suitablerepertoire of polypeptides or peptides (e.g., in a solution, immobilizedon a suitable support). The display system can also be a system thatemploys a cellular expression system (e.g., expression of a library ofnucleic acids in, e.g., transformed, infected, transfected or transducedcells and display of the encoded polypeptides on the surface of thecells) or an acellular expression system (e.g., emulsioncompartmentalization and display). Exemplary display systems link thecoding function of a nucleic acid and physical, chemical and/orfunctional characteristics of a polypeptide or peptide encoded by thenucleic acid. When such a display system is employed, polypeptides orpeptides that have a desired physical, chemical and/or functionalcharacteristic can be selected and a nucleic acid encoding the selectedpolypeptide or peptide can be readily isolated or recovered. A number ofdisplay systems that link the coding function of a nucleic acid andphysical, chemical and/or functional characteristics of a polypeptide orpeptide are known in the art, for example, bacteriophage display (phagedisplay, for example phagemid display), ribosome display, emulsioncompartmentalization and display, yeast display, puromycin display,bacterial display, display on plasmid, covalent display and the like.(See, e.g., EP 0436597 (Dyax), U.S. Pat. No. 6,172,197 (McCafferty etal.), U.S. Pat. No. 6,489,103 (Griffiths et al.).)

As used herein, “repertoire” refers to a collection of polypeptides orpeptides that are characterized by amino acid sequence diversity. Theindividual members of a repertoire can have common features, such ascommon structural features (e.g., a common core structure) and/or commonfunctional features (e.g., capacity to bind a common ligand (e.g., ageneric ligand or a target ligand, TNFR1)).

As used herein, “functional” describes a polypeptide or peptide that hasbiological activity, such as specific binding activity. For example, theterm “functional polypeptide” includes an antibody or antigen-bindingfragment thereof that binds a target antigen through its antigen-bindingsite.

As used herein, “generic ligand” refers to a ligand that binds asubstantial portion (e.g., substantially all) of the functional membersof a given repertoire. A generic ligand (e.g., a common generic ligand)can bind many members of a given repertoire even though the members maynot have binding specificity for a common target ligand. In general, thepresence of a functional generic ligand-binding site on a polypeptide(as indicated by the ability to bind a generic ligand) indicates thatthe polypeptide is correctly folded and functional. Suitable examples ofgeneric ligands include superantigens, antibodies that bind an epitopeexpressed on a substantial portion of functional members of arepertoire, and the like.

“Superantigen” is a term of art that refers to generic ligands thatinteract with members of the immunoglobulin superfamily at a site thatis distinct from the target ligand-binding sites of these proteins.Staphylococcal enterotoxins are examples of superantigens which interactwith T-cell receptors. Superantigens that bind antibodies includeProtein G, which binds the IgG constant region (Bjorck and Kronvall, J.Immunol., 133:969 (1984)); Protein A which binds the IgG constant regionand V_(H) domains (Forsgren and Sjoquist, J. Immunol., 97:822 (1966));and Protein L which binds V_(L) domains (Bjorck, J. Immunol., 140:1194(1988)).

As used herein, “target ligand” refers to a ligand which is specificallyor selectively bound by a polypeptide or peptide. For example, when apolypeptide is an antibody or antigen-binding fragment thereof, thetarget ligand can be any desired antigen or epitope. Binding to thetarget antigen is dependent upon the polypeptide or peptide beingfunctional.

As used herein an antibody refers to IgG, IgM, IgA, IgD or IgE or afragment (such as a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, closedconformation multispecific antibody, disulphide-linked scFv, diabody)whether derived from any species naturally producing an antibody, orcreated by recombinant DNA technology; whether isolated from serum,B-cells, hybridomas, transfectomas, yeast or bacteria.

As used herein, “antibody format” refers to any suitable polypeptidestructure in which one or more antibody variable domains can beincorporated so as to confer binding specificity for antigen on thestructure. A variety of suitable antibody formats are known in the art,such as, chimeric antibodies, humanized antibodies, human antibodies,single chain antibodies, bispecific antibodies, antibody heavy chains,antibody light chains, homodimers and heterodimers of antibody heavychains and/or light chains, antigen-binding fragments of any of theforegoing (e.g., a Fv fragment (e.g., single chain Fv (scFv), adisulfide bonded Fv), a Fab fragment, a Fab′ fragment, a F(ab′)₂fragment), a single antibody variable domain (e.g., a DAB™, V_(H),V_(HH), V_(L)), and modified versions of any of the foregoing (e.g.,modified by the covalent attachment of polyethylene glycol or othersuitable polymer or a humanized V_(HH)).

The phrase “immunoglobulin single variable domain” refers to an antibodyvariable domain (V_(H), V_(HH), V_(L)) that specifically binds anantigen or epitope independently of other V regions or domains. Animmunoglobulin single variable domain can be present in a format (e.g.,homo- or hetero-multimer) with other variable regions or variabledomains where the other regions or domains are not required for antigenbinding by the single immunoglobulin variable domain (i.e., where theimmunoglobulin single variable domain binds antigen independently of theadditional variable domains). A “domain antibody” or “DAB™” is the sameas an “immunoglobulin single variable domain” as the term is usedherein. A “single immunoglobulin variable domain” is the same as an“immunoglobulin single variable domain” as the term is used herein. A“single antibody variable domain” or an “antibody single variabledomain” is the same as an “immunoglobulin single variable domain” as theterm is used herein. An immunoglobulin single variable domain is in oneembodiment a human antibody variable domain, but also includes singleantibody variable domains from other species such as rodent (forexample, as disclosed in WO 00/29004, the contents of which areincorporated herein by reference in their entirety), nurse shark andCamelid V_(HH) DAB™s. Camelid V_(HH) are immunoglobulin single variabledomain polypeptides that are derived from species including camel,llama, alpaca, dromedary, and guanaco, which produce heavy chainantibodies naturally devoid of light chains. The V_(HH) may behumanized.

A “domain” is a folded protein structure which has tertiary structureindependent of the rest of the protein. Generally, domains areresponsible for discrete functional properties of proteins, and in manycases may be added, removed or transferred to other proteins withoutloss of function of the remainder of the protein and/or of the domain. A“single antibody variable domain” is a folded polypeptide domaincomprising sequences characteristic of antibody variable domains. Ittherefore includes complete antibody variable domains and modifiedvariable domains, for example, in which one or more loops have beenreplaced by sequences which are not characteristic of antibody variabledomains, or antibody variable domains which have been truncated orcomprise N- or C-terminal extensions, as well as folded fragments ofvariable domains which retain at least the binding activity andspecificity of the full-length domain.

The term “library” refers to a mixture of heterogeneous polypeptides ornucleic acids. The library is composed of members, each of which has asingle polypeptide or nucleic acid sequence. To this extent, “library”is synonymous with “repertoire.” Sequence differences between librarymembers are responsible for the diversity present in the library. Thelibrary may take the form of a simple mixture of polypeptides or nucleicacids, or may be in the form of organisms or cells, for examplebacteria, viruses, animal or plant cells and the like, transformed witha library of nucleic acids. In one embodiment, each individual organismor cell contains only one or a limited number of library members. In oneembodiment, the nucleic acids are incorporated into expression vectors,in order to allow expression of the polypeptides encoded by the nucleicacids. In an aspect, therefore, a library may take the form of apopulation of host organisms, each organism containing one or morecopies of an expression vector containing a single member of the libraryin nucleic acid form which can be expressed to produce its correspondingpolypeptide member. Thus, the population of host organisms has thepotential to encode a large repertoire of diverse polypeptides.

A “universal framework” is a single antibody framework sequencecorresponding to the regions of an antibody conserved in sequence asdefined by Kabat (“Sequences of Proteins of Immunological Interest”, USDepartment of Health and Human Services) or corresponding to the humangermline immunoglobulin repertoire or structure as defined by Chothiaand Lesk, (1987) J. Mol. Biol. 196:910-917. Libraries and repertoirescan use a single framework, or a set of such frameworks, which has beenfound to permit the derivation of virtually any binding specificitythough variation in the hypervariable regions alone.

As used herein, the term “dose” refers to the quantity of ligandadministered to a subject all at one time (unit dose), or in two or moreadministrations over a defined time interval. For example, dose canrefer to the quantity of ligand (e.g., ligand comprising animmunoglobulin single variable domain that binds target antigen)administered to a subject over the course of one day (24 hours) (dailydose), two days, one week, two weeks, three weeks or one or more months(e.g., by a single administration, or by two or more administrations).The interval between doses can be any desired amount of time.

The phrase, “half-life,” refers to the time taken for the serumconcentration of the ligand (e.g., DAB™, polypeptide or antagonist) toreduce by 50%, in vivo, for example due to degradation of the ligandand/or clearance or sequestration of the ligand by natural mechanisms.The ligands of the invention may be stabilized in vivo and theirhalf-life increased by binding to molecules which resist degradationand/or clearance or sequestration. Typically, such molecules arenaturally occurring proteins which themselves have a long half-life invivo. The half-life of a ligand is increased if its functional activitypersists, in vivo, for a longer period than a similar ligand which isnot specific for the half-life increasing molecule. For example, aligand specific for human serum albumin (HAS) and a target molecule iscompared with the same ligand wherein the specificity to HSA is notpresent, that is does not bind HSA but binds another molecule. Forexample, it may bind a third target on the cell. Typically, thehalf-life is increased by 10%, 20%, 30%, 40%, 50% or more. Increases inthe range of 2×, 3×, 4×, 5×, 10×, 20×, 30×, 40×, 50× or more of thehalf-life are possible. Alternatively, or in addition, increases in therange of up to 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 150× of thehalf-life are possible.

As used herein, “hydrodynamic size” refers to the apparent size of amolecule (e.g., a protein molecule, ligand) based on the diffusion ofthe molecule through an aqueous solution. The diffusion, or motion of aprotein through solution can be processed to derive an apparent size ofthe protein, where the size is given by the “Stokes radius” or“hydrodynamic radius” of the protein particle. The “hydrodynamic size”of a protein depends on both mass and shape (conformation), such thattwo proteins having the same molecular mass may have differinghydrodynamic sizes based on the overall conformation of the protein.

As referred to herein, the term “competes” means that the binding of afirst target to its cognate target binding domain is inhibited in thepresence of a second binding domain that is specific for said cognatetarget. For example, binding may be inhibited sterically, for example byphysical blocking of a binding domain or by alteration of the structureor environment of a binding domain such that its affinity or avidity fora target is reduced. See WO2006038027 for details of how to performcompetition ELISA and competition BIACORE™ experiments to determinecompetition between first and second binding domains.

Calculations of “homology” or “identity” or “similarity” between twosequences (the terms are used interchangeably herein) are performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Inan embodiment, the length of a reference sequence aligned for comparisonpurposes is at least 30%, or at least 40%, or at least 50%, or at least60%, or at least 70%, 80%, 90%, 100% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “homology” is equivalent to amino acidor nucleic acid “identity”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences. Amino acid and nucleotide sequence alignments and homology,similarity or identity, as defined herein may be prepared and determinedusing the algorithm BLAST 2 Sequences, using default parameters(Tatusova, T. A. et al., FEMS Microbiol Lett, 174:187-188 (1999)).

Selection Methods

The invention in one embodiment relates to polypeptides and DAB™sselected by a method of selection of protease resistant peptides andpolypeptides that have a desired biological activity. Two selectivepressures are used in the method to produce an efficient process forselecting polypeptides that are highly stable and resistant to proteasedegradation, and that have desired biological activity. As describedherein, protease resistant peptides and polypeptides generally retainbiological activity. In contrast, protease sensitive peptides andpolypeptides are cleaved or digested by protease in the methodsdescribed herein, and therefore, lose their biological activity.Accordingly, protease resistant peptides or polypeptides are generallyselected based on their biological activity, such as binding activity.

The methods described herein provide several advantages. For example, asdisclosed and exemplified herein, variable domains, antagonists,peptides or polypeptides that are selected for resistance to proteolyticdegradation by one protease (e.g., trypsin), are also resistant todegradation by other proteases (e.g., elastase, leucozyme). In oneembodiment protease resistance correlates with a higher meltingtemperature (Tm) of the peptide or polypeptide. Higher meltingtemperatures are indicative of more stable variable domains,antagonists, peptides and polypeptides. Resistance to proteasedegradation also correlates in one embodiment with high affinity bindingto target ligands. Thus, the methods described herein provide anefficient way to select, isolate and/or recover variable domains,antagonists, peptides, polypeptides that have a desired biologicalactivity and that are well suited for in vivo therapeutic and/ordiagnostic uses because they are protease resistant and stable. In oneembodiment protease resistance correlates with an improved PK, forexample improved over n variable domain, antagonist, peptide orpolypeptide that is not protease resistant. Improved PK may be animproved AUC (area under the curve) and/or an improved half-life. In oneembodiment protease resistance correlates with an improved stability ofthe variable domain, antagonist, peptide or polypeptide to shear and/orthermal stress and/or a reduced propensity to aggregate duringnebulisation, for example improved over an variable domain, antagonist,peptide or polypeptide that is not protease resistant. In one embodimentprotease resistance correlates with an improved storage stability, forexample improved over an variable domain, antagonist, peptide orpolypeptide that is not protease resistant. In one aspect, one, two,three, four or all of the advantages are provided, the advantages beingresistance to protease degradation, higher Tm and high affinity bindingto target ligand.

In one aspect, there is provided a method for selecting, isolatingand/or recovering a peptide or polypeptide from a library or arepertoire of peptides and polypeptides (e.g., a display system) that isresistant to degradation by a protease (e.g., one or more proteases). Inone embodiment, the method is a method for selecting, isolating and/orrecovering a polypeptide from a library or a repertoire of peptides andpolypeptides (e.g., a display system) that is resistant to degradationby a protease (e.g., one or more proteases). Generally, the methodcomprises providing a library or repertoire of peptides or polypeptides,combining the library or repertoire with a protease (e.g., trypsin,elastase, leucozyme, pancreatin, sputum) under conditions suitable forprotease activity, and selecting, isolating and/or recovering a peptideor polypeptide that is resistant to degradation by the protease and hasa desired biological activity. Peptides or polypeptides that aredegraded by a protease generally have reduced biological activity orlose their biological activity due to the activity of protease.Accordingly, peptides or polypeptides that are resistant to proteasedegradation can be selected, isolated and/or recovered using the methodbased on their biological activity, such as binding activity (e.g.,binding a general ligand, binding a specific ligand, binding asubstrate), catalytic activity or other biological activity.

The library or repertoire of peptides or polypeptides is combined with aprotease (e.g., one or more proteases) under conditions suitable forproteolytic activity of the protease. Conditions that are suitable forproteolytic activity of protease, and biological preparations ormixtures that contain proteolytic activity, are well-known in the art orcan be readily determined by a person of ordinary skill in the art. Ifdesired, suitable conditions can be identified or optimized, forexample, by assessing protease activity under a range of pH conditions,protease concentrations, temperatures and/or by varying the amount oftime the library or repertoire and the protease are permitted to react.For example, in some embodiments, the ratio (on a mole/mole basis) ofprotease, e.g. trypsin, to peptide or polypeptide (e.g., variabledomain) is 800 to 80,00 (e.g., 8,000 to 80,000) protease:peptide orpolypeptide, e.g. when 10 micrograms/ml of protease is used, the ratiois 800 to 80,000 protease:peptide or polypeptide; or when 100micrograms/ml of protease is used, the ratio is 8,000 to 80,000protease:peptide or polypeptide. In one embodiment the ratio (on aweight/weight, e.g. microgram/microgram basis) of protease (e.g.,trypsin) to peptide or polypeptide (e.g., variable domain) is 1,600 to160,000 (e.g., 16,000 to 160,000) protease:peptide or polypeptide e.g.when 10 micrograms/ml of protease is used, the ratio is 1,600 to 160,000protease:peptide or polypeptide; or when 100 micrograms/ml of proteaseis used, the ratio is 16,000 to 160,000 protease:peptide or polypeptide.In one embodiment, the protease is used at a concentration of at least100 or 1000 micrograms/ml and the protease:peptide ratio (on a mole/molebasis) of protease, e.g. trypsin, to peptide or polypeptide (e.g.,variable domain)

is 8,000 to 80,000 protease:peptide or polypeptide. In one embodiment,the protease is used at a concentration of at least 10 micrograms/ml andthe protease:peptide ratio (on a mole/mole basis) of protease, e.g.trypsin, to peptide or polypeptide (e.g., variable domain) is 800 to80,000 protease:peptide or polypeptide. In one embodiment the ratio (ona weight/weight, e.g. microgram/microgram basis) of protease (e.g.,trypsin) to peptide or polypeptide (e.g., variable domain) is 1600 to160,000 protease:peptide or polypeptide e.g. when C is 10 micrograms/ml;or when C or C′ is 100 micrograms/ml, the ratio is 16,000 to 160,000protease:peptide or polypeptide. In one embodiment, the concentration (cor c′) is at least 100 or 1000 micrograms/ml protease. For testing anindividual or isolated peptide or polypeptide (e.g., an immunoglobulinvariable domain), e.g. one that has already been isolated from arepertoire or library, a protease can be added to a solution of peptideor polypeptide in a suitable buffer (e.g., PBS) to produce a peptide orpolypeptide/protease solution, such as a solution of at least about0.01% (w/w) protease/peptide or polypeptide, about 0.01% to about 5%(w/w) protease/peptide or polypeptide, about 0.05% to about 5% (w/w)protease/peptide or polypeptide, about 0.1% to about 5% (w/w)protease/peptide or polypeptide, about 0.5% to about 5% (w/w)protease/peptide or polypeptide, about 1% to about 5% (w/w)protease/peptide or polypeptide, at least about 0.01% (w/w)protease/peptide or polypeptide, at least about 0.02% (w/w)protease/peptide or polypeptide, at least about 0.03% (w/w)protease/peptide or polypeptide, at least about 0.04% (w/w)protease/peptide or polypeptide, at least about 0.05% (w/w)protease/peptide or polypeptide, at least about 0.06% (w/w)protease/peptide or polypeptide, at least about 0.07% (w/w)protease/peptide or polypeptide, at least about 0.08% (w/w)protease/peptide or polypeptide, at least about 0.09% (w/w)protease/peptide or polypeptide, at least about 0.1% (w/w)protease/peptide or polypeptide, at least about 0.2% (w/w)protease/peptide or polypeptide, at least about 0.3% (w/w)protease/peptide or polypeptide, at least about 0.4% (w/w)protease/peptide or polypeptide, at least about 0.5% (w/w)protease/peptide or polypeptide, at least about 0.6% (w/w)protease/peptide or polypeptide, at least about 0.7% (w/w)protease/peptide or polypeptide, at least about 0.8% (w/w)protease/peptide or polypeptide, at least about 0.9% (w/w)protease/peptide or polypeptide, at least about 1% (w/w)protease/peptide or polypeptide, at least about 2% (w/w)protease/peptide or polypeptide, at least about 3% (w/w)protease/peptide or polypeptide, at least about 4% (w/w)protease/peptide or polypeptide, or about 5% (w/w) protease/peptide orpolypeptide. The mixture can be incubated at a suitable temperature forprotease activity (e.g., room temperature, about 37° C.) and samples canbe taken at time intervals (e.g., at 1 hour, 2 hours, 3 hours, etc.).The samples can be analyzed for protein degradation using any suitablemethod, such as SDS-PAGE analysis or ligand binding, and the results canbe used to establish a time course of degradation.

Any desired protease or proteases can be used in the methods describedherein. For example, a single protease, any desired combination ofdifferent proteases, or any biological preparation, biological extract,or biological homogenate that contains proteolytic activity can be used.It is not necessary that the identity of the protease or proteases thatare used be known. Suitable examples of proteases that can be used aloneor in any desired combination include serine protease, cysteineprotease, aspartate proteases, thiol proteases, matrix metalloprotease,carboxypeptidase (e.g., carboxypeptidase A, carboxypeptidase B),trypsin, chymotrypsin, pepsin, papain, elastase, leukozyme, pancreatin,thrombin, plasmin, cathepsins (e.g., cathepsin G), proteinase (e.g.,proteinase 1, proteinase 2, proteinase 3), thermolysin, chymosin,enteropeptidase, caspase (e.g., caspase 1, caspase 2, caspase 4, caspase5, caspase 9, caspase 12, caspase 13), calpain, ficain, clostripain,actinidain, bromelain, separase and the like. Suitable biologicalextracts, homogenates and preparations that contains proteolyticactivity include sputum, mucus (e.g., gastric mucus, nasal mucus,bronchial mucus), bronchoalveolar lavage, lung homogenate, lung extract,pancreatic extract, gastric fluid, saliva, tears and the like. Theprotease is used in an amount suitable for proteolytic degradation tooccur. For example, as described herein, protease can be used at about0.01% to about 5% (w/w, protease/peptide or polypeptide). When proteaseis combined with a display system that comprises the repertoire ofpeptides or polypeptides (e.g., a phage display system), for example,the protease can be used at a concentration of about 10 μg/ml to about 3mg/ml, about 10 μg/ml, about 20 μg/ml, about 30 μg/ml, about 40 μg/ml,about 50 μg/ml, about 60 μg/ml, about 70 μg/ml, about 80 μg/ml, about 90μg/ml, about 100 μg/ml, about 200 μg/ml, about 300 μg/ml, about 400μg/ml, about 500 μg/ml, about 600 μg/ml, about 700 μg/ml, about 800μg/ml, about 900 μg/ml, about 1000 μg/ml, about 1.5 mg/ml, about 2mg/ml, about 2.5 mg/ml or about 3 mg/ml.

The protease is incubated with the collection of peptides orpolypeptides (library or repertoire) at a temperature that is suitablefor activity of the protease. For example, the protease and collectionof peptides or polypeptides can be incubated at a temperature of about20° C. to about 40° C. (e.g., at room temperature, about 20° C., about21° C., about 22° C., about 23° C., about 24° C., about 25° C., about26° C., about 27° C., about 28° C., about 29° C., about 30° C., about31° C., about 32° C., about 33° C., about 34° C., about 35° C., about36° C., about 37° C., about 38° C., about 39° C., about 40° C.). Theprotease and the collection of peptides or polypeptides are incubatedtogether for a period of time sufficient for proteolytic degradation tooccur. For example, the collection of peptides or polypeptides can beincubated together with protease for about 30 minutes to about 24 orabout 48 hours. In some examples, the collection of peptides orpolypeptides is incubated together with protease overnight, or for atleast about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours,about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours,about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours,about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 48hours, or longer.

It is generally desirable, at least in early selection rounds (e.g. whena display system is used), that the protease results in a reduction inthe number of clones that have the desired biological activity that isselected for by at least one order of magnitude, in comparison toselections that do not include incubation with protease. In particularexamples, the amount of protease and conditions used in the methods aresufficient to reduce the number of recovered clones by at least aboutone log (a factor of 10), at least about 2 logs (a factor of 100), atleast about 3 logs (a factor of 1000) or at least about 4 logs (a factorof 10,000). Suitable amounts of protease and incubation conditions thatwill result in the desired reduction in recovered clones can be easilydetermined using conventional methods and/or the guidance providedherein.

The protease and collection of peptides or polypeptides can be combinedand incubated using any suitable method (e.g., in vitro, in vivo or exvivo). For example, the protease and collection of peptides orpolypeptides can be combined in a suitable container and heldstationary, rocked, shaken, swirled or the like, at a temperaturesuitable for protease activity. If desired, the protease and collectionof peptides or polypeptides can be combined in an in vivo or ex vivosystem, such as by introducing the collection of polypeptides (e.g., aphage display library or repertoire) into a suitable animal (e.g., amouse), and after sufficient time for protease activity has passed,recovering the collection of peptides or polypeptides. In anotherexample, an organ or tissue is perfused with the collection ofpolypeptides (e.g., a phage display library or repertoire), and aftersufficient time for protease activity has passed, the collection ofpolypeptides is recovered.

Following incubation, a protease resistant peptide or polypeptide can beselected based on a desired biological activity, such as a bindingactivity. If desired, a protease inhibitor can be added beforeselection. Any suitable protease inhibitor (or combination of two ormore protease inhibitors) that will not substantially interfere with theselection method can be used. Examples of suitable protease inhibitorsinclude, α1-anti-trypsin, α2-macroglobulin, amastatin, antipain,antithrombin III, aprotinin, 4-(2-Aminoethyl)benzenesulfonyl fluoridehydrochloride (AEBSF), (4-amidino-phenyl)-methane-sulfonyl fluoride(APMSF), bestatin, benzamidine, chymostatin, 3,4-dichloroisocoumarin,diisoproply fluorophosphate (DIFP), E-64, ethylenediamine tetraacedicacid (EDTA), elastatinal, leupeptin, N-ethylmaleimide,phenylmethylsulfonylfluoride (PMSF), pepstatin, 1,10-phenanthroline,phosphoramidon, serine protease inhibitors,N-tosyl-L-lysine-chloromethyl ketone (TLCK),Na-tosyl-Phe-chloromethylketone (TPCK) and the like. In addition, manypreparations that contain inhibitors of several classes of proteases arecommercially available (e.g., ROCHE COMPLETE PROTEASE INHIBITOR COCKTAILTABLETS™ (Roche Diagnostics Corporation; Indianapolis, Ind., USA), whichinhibits chymotrypsin, thermolysin, papain, pronase, pancreatic extractand trypsin).

A protease resistant peptide or polypeptide can be selected using adesired biological activity selection method, which allows peptides andpolypeptides that have the desired biological activity to bedistinguished from and selected over peptides and polypeptides that donot have the desired biological activity. Generally, peptides orpolypeptides that have been digested or cleaved by protease loose theirbiological activity, while protease resistant peptides or polypeptidesremain functional. Thus, suitable assays for biological activity can beused to select protease resistant peptides or polypeptides. For example,a common binding function (e.g., binding of a general ligand, binding ofa specific ligand, or binding of a substrate) can be assessed using asuitable binding assay (e.g., ELISA, panning). For example, polypeptidesthat bind a target ligand or a generic ligand, such as protein A,protein L or an antibody, can be selected, isolated, and/or recovered bypanning or using a suitable affinity matrix. Panning can be accomplishedby adding a solution of ligand (e.g., generic ligand, target ligand) toa suitable vessel (e.g., tube, petri dish) and allowing the ligand tobecome deposited or coated onto the walls of the vessel. Excess ligandcan be washed away and polypeptides (e.g., a phage display library) canbe added to the vessel and the vessel maintained under conditionssuitable for the polypeptides to bind the immobilized ligand. Unboundpolypeptide can be washed away and bound polypeptides can be recoveredusing any suitable method, such as scraping or lowering the pH, forexample.

When a phage display system is used, binding can be tested in a phageELISA. Phage ELISA may be performed according to any suitable procedure.In one example, populations of phage produced at each round of selectioncan be screened for binding by ELISA to the selected target ligand orgeneric ligand, to identify phage that display protease resistantpeptides or polypeptides. If desired, soluble peptides and polypeptidescan be tested for binding to target ligand or generic ligand, forexample by ELISA using reagents, for example, against a C- or N-terminaltag (see for example Winter et al. (1994) Ann. Rev. Immunology 12,433-55 and references cited therein). The diversity of the selectedphage may also be assessed by gel electrophoresis of PCR products (Markset al. 1991, supra; Nissim et al. 1994 supra), probing (Tomlinson etal., 1992) J. Mol. Biol. 227, 776) or by sequencing of the vector DNA.

In addition to specificity for TNFR1, an antagonist or polypeptide(e.g., a dual specific ligand) comprising an anti-TNFR1 proteaseresistant polypeptide (e.g., single antibody variable domain) can havebinding specificity for a generic ligand or any desired target ligand,such as human or animal proteins, including cytokines, growth factors,cytokine receptors, growth factor receptors, enzymes (e.g., proteases),co-factors for enzymes, DNA binding proteins, lipids and carbohydrates.

In some embodiments, the protease resistant peptide or polypeptide(e.g., DAB™) or antagonist binds TNFR1 in pulmonary tissue. In oneembodiment, the antagonist or polypeptide also binds a further target inpulmonary tissue.

When a display system (e.g., a display system that links coding functionof a nucleic acid and functional characteristics of the peptide orpolypeptide encoded by the nucleic acid) is used in the methodsdescribed herein it may be frequently advantageous to amplify orincrease the copy number of the nucleic acids that encode the selectedpeptides or polypeptides. This provides an efficient way of obtainingsufficient quantities of nucleic acids and/or peptides or polypeptidesfor additional rounds of selection, using the methods described hereinor other suitable methods, or for preparing additional repertoires(e.g., affinity maturation repertoires). Thus, in some embodiments, themethods comprise using a display system (e.g., that links codingfunction of a nucleic acid and functional characteristics of the peptideor polypeptide encoded by the nucleic acid, such as phage display) andfurther comprises amplifying or increasing the copy number of a nucleicacid that encodes a selected peptide or polypeptide. Nucleic acids canbe amplified using any suitable methods, such as by phage amplification,cell growth or polymerase chain reaction.

The methods described herein can be used as part of a program to isolateprotease resistant peptides or polypeptides, e.g. DAB™s, that cancomprise, if desired, other suitable selection methods. In thesesituations, the methods described herein can be employed at any desiredpoint in the program, such as before or after other selection methodsare used. The methods described herein can also be used to provide twoor more rounds of selection, as described and exemplified herein.

In one example, the method is for selecting a peptide or polypeptide,e.g. a DAB™, that is resistant to degradation by elastase, comprisingproviding a library or repertoire of peptides or polypeptides, combiningthe library or repertoire with elastase (or a biological preparation,extract or homogenate comprising elastase) under conditions suitable forproteolytic digestion by elastase, and selecting, isolating and/orrecovering a peptide or polypeptide that is resistant to degradation byelastase and has TNFR1 binding activity.

In particular embodiments, there is provided a method for selecting animmunoglobulin single variable domain (a DAB™) that is resistant todegradation by elastase and binds TNFR1. In these embodiments, a libraryor repertoire comprising DAB™s is provided and combined with elastase(or a biological preparation, extract or homogenate comprising elastase)under conditions suitable for proteolytic digestion by elastase.Elastase resistant DAB™s are selected that specifically bind TNFR1. Forexample, the elastase resistant DAB™ is not substantially degraded whenincubated at 37° C. in a 0.04% (w/w) solution of elastase for a periodof at least about 2 hours. In one embodiment, the elastase resistantDAB™ is not substantially degraded when incubated at 37° C. in a 0.04%(w/w) solution of elastase for a period of at least about 12 hours. Inone embodiment, the elastase resistant DAB™ is not substantiallydegraded when incubated at 37° C. in a 0.04% (w/w) solution of elastasefor a period of at least about 24 hours, at least about 36 hours, or atleast about 48 hours.

In an embodiment, there is provided a method for selecting animmunoglobulin single variable domain (a DAB™) that is resistant todegradation by elastase and binds TNFR1. The method comprises providinga phage display system comprising a repertoire of polypeptides thatcomprise an immunoglobulin single variable domain, combining the phagedisplay system with elastase (about 100 μg/ml) and incubating themixture at about 37° C., for example, overnight (e.g., about 12-16hours), and then selecting phage that display a DAB™ that specificallybinds TNFR1.

In one example, there is provided a method for selecting a peptide orpolypeptide (e.g., a DAB™) that is resistant to degradation byleucozyme, comprising providing a library or repertoire of peptides orpolypeptides, combining the library or repertoire with leucozyme (or abiological preparation, extract or homogenate comprising leucozyme)under conditions suitable for proteolytic digestion by leucozyme, andselecting, isolating and/or recovering a peptide or polypeptide that isresistant to degradation by leucozyme and has specific TNFR1 bindingactivity.

In particular embodiments, there is provided a method for selecting animmunoglobulin single variable domain (a DAB™) that is resistant todegradation by leucozyme and binds TNFR1. In these embodiments, alibrary or repertoire comprising DAB™s is provided and combined withleucozyme (or a biological preparation, extract or homogenate comprisingleucozyme) under conditions suitable for proteolytic digestion byleucozyme. Leucozyme resistant DAB™s are selected that specifically bindTNFR1. For example, the leucozyme resistant DAB™ is not substantiallydegraded when incubated at 37° C. in a 0.04% (w/w) solution of leucozymefor a period of at least about 2 hours. In one embodiment, the leucozymeresistant DAB™ is not substantially degraded when incubated at 37° C. ina 0.04% (w/w) solution of leucozyme for a period of at least about 12hours. In one embodiment, the leucozyme resistant DAB™ is notsubstantially degraded when incubated at 37° C. in a 0.04% (w/w)solution of leucozyme for a period of at least about 24 hours, at leastabout 36 hours, or at least about 48 hours.

In an embodiment, there is provided a method for selecting animmunoglobulin single variable domain (a DAB™) that is resistant todegradation by leucozyme and specifically binds TNFR1. The methodcomprises providing a phage display system comprising a repertoire ofpolypeptides that comprise an immunoglobulin single variable domain,combining the phage display system with leucozyme (about 100 μg/ml) andincubating the mixture at about 37° C., for example, overnight (e.g.,about 12-16 hours), and then selecting phage that display a DAB™ thatspecifically bind TNFR1.

In another example, there is provided a method for selecting a peptideor polypeptide (e.g., a DAB™) that is resistant to degradation bytrypsin, comprising providing a library or repertoire of peptides orpolypeptides, combining the library or repertoire with trypsin underconditions suitable for proteolytic digestion by trypsin, and selecting,isolating and/or recovering a peptide or polypeptide that is resistantto degradation by trypsin and specifically binds TNFR1.

In particular embodiments, there is provided a method for selecting animmunoglobulin single variable domain (a DAB™) that is resistant todegradation by trypsin and specifically binds TNFR1. In theseembodiments, a library or repertoire comprising DAB™s is provided andcombined with trypsin (or a biological preparation, extract orhomogenate comprising trypsin) under conditions suitable for proteolyticdigestion by trypsin. Trypsin resistant DAB™s are selected that bindTNFR1. For example, the trypsin resistant DAB™ is not substantiallydegraded when incubated at 37° C. in a 0.04% (w/w) solution of trypsinfor a period of at least about 2 hours. In one embodiment, the trypsinresistant DAB™ is not substantially degraded when incubated at 37° C. ina 0.04% (w/w) solution of trypsin for a period of at least about 3hours. In one embodiment, the trypsin resistant DAB™ is notsubstantially degraded when incubated at 37° C. in a 0.04% (w/w)solution of trypsin for a period of at least about 4 hours, at leastabout 5 hours, at least about 6 hours, at least about 7 hours, at leastabout 8 hours, at least about 9 hours, at least about 10 hours, at leastabout 11 hours, or at least about 12 hours.

In an exemplary embodiment, there is provided a method for selecting animmunoglobulin single variable domain (a DAB™) that is resistant todegradation by trypsin and specifically binds TNFR1. The methodcomprises providing a phage display system comprising a repertoire ofpolypeptides that comprise an immunoglobulin single variable domain,combining the phage display system with trypsin (100 μg/ml) andincubating the mixture at about 37° C., for example overnight (e.g.,about 12-16 hours), and then selecting phage that display a DAB™ thatspecifically bind TNFR1.

In another aspect, there is provided a method of producing a repertoireof protease resistant peptides or polypeptides (e.g., DAB™s). The methodcomprises providing a repertoire of peptides or polypeptides; combiningthe repertoire of peptides or polypeptides and a protease under suitableconditions for protease activity; and recovering a plurality of peptidesor polypeptides that specifically bind TNFR1, whereby a repertoire ofprotease resistant peptides or polypeptides is produced. Proteases,display systems, conditions for protease activity, and methods forselecting peptides or polypeptides that are suitable for use in themethod are described herein with respect to the other methods.

In some embodiments, a display system (e.g., a display system that linkscoding function of a nucleic acid and functional characteristics of thepeptide or polypeptide encoded by the nucleic acid) that comprises arepertoire of peptides or polypeptides is used, and the method furthercomprises amplifying or increasing the copy number of the nucleic acidsthat encode the plurality of selected peptides or polypeptides. Nucleicacids can be amplified using any suitable method, such as by phageamplification, cell growth or polymerase chain reaction.

In particular embodiment, there is provided a method of producing arepertoire of protease resistant polypeptides that comprise anti-TNFR1DAB™s. The method comprises providing a repertoire of polypeptides thatcomprise DAB™s; combining the repertoire of peptides or polypeptides anda protease (e.g., trypsin, elastase, leucozyme) under suitableconditions for protease activity; and recovering a plurality ofpolypeptides that comprise DAB™s that have binding specificity forTNFR1. The method can be used to produce a naïve repertoire, or arepertoire that is biased toward a desired binding specificity, such asan affinity maturation repertoire based on a parental DAB™ that hasbinding specificity for TNFR1.

Polypeptide Display Systems

In one embodiment, the repertoire or library of peptides or polypeptidesprovided for use in the methods described herein comprise a suitabledisplay system. The display system may resist degradation by protease(e.g., a single protease or a combination of proteases, and anybiological extract, homogenate or preparation that contains proteolyticactivity (e.g., sputum, mucus (e.g., gastric mucus, nasal mucus,bronchial mucus), bronchoalveolar lavage, lung homogenate, lung extract,pancreatic extract, gastric fluid, saliva, tears and the like). Thedisplay system and the link between the display system and the displayedpolypeptide is in one embodiment at least as resistant to protease asthe most stable peptides or polypeptides of the repertoire. This allowsa nucleic acid that encodes a selected displayed polypeptide to beeasily isolated and/or amplified.

In one example, a protease resistant peptide or polypeptide, e.g. aDAB™, can be selected, isolated and/or recovered from a repertoire ofpeptides or polypeptides that is in solution, or is covalently ornoncovalently attached to a suitable surface, such as plastic or glass(e.g., microtiter plate, polypeptide array such as a microarray). Forexample an array of peptides on a surface in a manner that places eachdistinct library member (e.g., unique peptide sequence) at a discrete,predefined location in the array can be used. The identity of eachlibrary member in such an array can be determined by its spatiallocation in the array. The locations in the array where bindinginteractions between a target ligand, for example, and reactive librarymembers occur can be determined, thereby identifying the sequences ofthe reactive members on the basis of spatial location. (See, e.g., U.S.Pat. No. 5,143,854, WO 90/15070 and WO 92/10092.)

In one embodiment, the methods employ a display system that links thecoding function of a nucleic acid and physical, chemical and/orfunctional characteristics of the polypeptide encoded by the nucleicacid. Such a display system can comprise a plurality of replicablegenetic packages, such as bacteriophage or cells (bacteria). In oneembodiment, the display system comprises a library, such as abacteriophage display library.

A number of suitable bacteriophage display systems (e.g., monovalentdisplay and multivalent display systems) have been described. (See,e.g., Griffiths et al., U.S. Pat. No. 6,555,313 B1 (incorporated hereinby reference); Johnson et al., U.S. Pat. No. 5,733,743 (incorporatedherein by reference); McCafferty et al., U.S. Pat. No. 5,969,108(incorporated herein by reference); Mulligan-Kehoe, U.S. Pat. No.5,702,892 (Incorporated herein by reference); Winter, G. et al., Annu.Rev. Immunol. 12:433-455 (1994); Soumillion, P. et al., Appl. Biochem.Biotechnol. 47(2-3):175-189 (1994); Castagnoli, L. et al., Comb. Chem.High Throughput Screen, 4(2):121-133 (2001).) The peptides orpolypeptides displayed in a bacteriophage display system can bedisplayed on any suitable bacteriophage, such as a filamentous phage(e.g., fd, M13, F1), a lytic phage (e.g., T4, T7, lambda), or an RNAphage (e.g., MS2), for example.

Generally, a library of phage that displays a repertoire of peptides orphage polypeptides, as fusion proteins with a suitable phage coatprotein (e.g., fd pIII protein), is produced or provided. The fusionprotein can display the peptides or polypeptides at the tip of the phagecoat protein, or if desired at an internal position. For example, thedisplayed peptide or polypeptide can be present at a position that isamino-terminal to domain 1 of pIII. (Domain 1 of pIII is also referredto as N1.) The displayed polypeptide can be directly fused to pIII(e.g., the N-terminus of domain 1 of pIII) or fused to pIII using alinker. If desired, the fusion can further comprise a tag (e.g., mycepitope, His tag). Libraries that comprise a repertoire of peptides orpolypeptides that are displayed as fusion proteins with a phage coatprotein can be produced using any suitable methods, such as byintroducing a library of phage vectors or phagemid vectors encoding thedisplayed peptides or polypeptides into suitable host bacteria, andculturing the resulting bacteria to produce phage (e.g., using asuitable helper phage or complementing plasmid if desired). The libraryof phage can be recovered from the culture using any suitable method,such as precipitation and centrifugation.

The display system can comprise a repertoire of peptides or polypeptidesthat contains any desired amount of diversity. For example, therepertoire can contain peptides or polypeptides that have amino acidsequences that correspond to naturally occurring polypeptides expressedby an organism, group of organisms (e.g., a repertoire of sequences ofV_(HH) DAB™s isolated from a Camelid), desired tissue or desired celltype, or can contain peptides or polypeptides that have random orrandomized amino acid sequences. If desired, the polypeptides can sharea common core or scaffold. The polypeptides in such a repertoire orlibrary can comprise defined regions of random or randomized amino acidsequence and regions of common amino acid sequence. In certainembodiments, all or substantially all polypeptides in a repertoire areof a desired type, such as a desired enzyme (e.g., a polymerase) or adesired antigen-binding fragment of an antibody (e.g., human V_(H) orhuman V_(L)). In embodiments, the polypeptide display system comprises arepertoire of polypeptides wherein each polypeptide comprises anantibody variable domain. For example, each polypeptide in therepertoire can contain a V_(H), a V_(L) or an Fv (e.g., a single chainFv).

Amino acid sequence diversity can be introduced into any desired regionof a peptide or polypeptide or scaffold using any suitable method. Forexample, amino acid sequence diversity can be introduced into a targetregion, such as a complementarity determining region of an antibodyvariable domain or a hydrophobic domain, by preparing a library ofnucleic acids that encode the diversified polypeptides using anysuitable mutagenesis methods (e.g., low fidelity PCR,oligonucleotide-mediated or site directed mutagenesis, diversificationusing NNK codons) or any other suitable method. If desired, a region ofa polypeptide to be diversified can be randomized.

The size of the polypeptides that make up the repertoire is largely amatter of choice and uniform polypeptide size is not required. In oneembodiment, the polypeptides in the repertoire have at least tertiarystructure (form at least one domain).

Selection/Isolation/Recovery

A protease resistant peptide or polypeptide (e.g., a population ofprotease resistant polypeptides) can be selected, isolated and/orrecovered from a repertoire or library (e.g., in a display system) usingany suitable method. In one embodiment, a protease resistant polypeptideis selected or isolated based on a selectable characteristic (e.g.,physical characteristic, chemical characteristic, functionalcharacteristic). Suitable selectable functional characteristics includebiological activities of the peptides or polypeptides in the repertoire,for example, binding to a generic ligand (e.g., a superantigen), bindingto a target ligand (e.g., an antigen, an epitope, a substrate), bindingto an antibody (e.g., through an epitope expressed on a peptide orpolypeptide), and catalytic activity. (See, e.g., Tomlinson et al., WO99/20749; WO 01/57065; WO 99/58655). In one embodiment, the selection isbased on specific binding to TNFR1. In another embodiment, selection ison the basis of the selected functional characteristic to produce asecond repertoire in which members are protease resistant, followed byselection of a member from the second repertoire that specifically bindsTNFR1.

In some embodiments, the protease resistant peptide or polypeptide isselected and/or isolated from a library or repertoire of peptides orpolypeptides in which substantially all protease resistant peptides orpolypeptides share a common selectable feature. For example, theprotease resistant peptide or polypeptide can be selected from a libraryor repertoire in which substantially all protease resistant peptides orpolypeptides bind a common generic ligand, bind a common target ligand,bind (or are bound by) a common antibody, or possess a common catalyticactivity. This type of selection is particularly useful for preparing arepertoire of protease resistant peptides or polypeptides that are basedon a parental peptide or polypeptide that has a desired biologicalactivity, for example, when performing affinity maturation of animmunoglobulin single variable domain.

Selection based on binding to a common generic ligand can yield acollection or population of peptides or polypeptides that contain all orsubstantially all of the protease resistant peptides or polypeptidesthat were components of the original library or repertoire. For example,peptides or polypeptides that bind a target ligand or a generic ligand,such as protein A, protein L or an antibody, can be selected, isolatedand/or recovered by panning or using a suitable affinity matrix. Panningcan be accomplished by adding a solution of ligand (e.g., genericligand, target ligand) to a suitable vessel (e.g., tube, petri dish) andallowing the ligand to become deposited or coated onto the walls of thevessel. Excess ligand can be washed away and peptides or polypeptides(e.g., a repertoire that has been incubated with protease) can be addedto the vessel and the vessel maintained under conditions suitable forpeptides or polypeptides to bind the immobilized ligand. Unboundpeptides or polypeptides can be washed away and bound peptides orpolypeptides can be recovered using any suitable method, such asscraping or lowering the pH, for example.

Suitable ligand affinity matrices generally contain a solid support orbead (e.g., agarose) to which a ligand is covalently or noncovalentlyattached. The affinity matrix can be combined with peptides orpolypeptides (e.g., a repertoire that has been incubated with protease)using a batch process, a column process or any other suitable processunder conditions suitable for binding of peptides or polypeptides to theligand on the matrix. Peptides or polypeptides that do not bind theaffinity matrix can be washed away and bound peptides or polypeptidescan be eluted and recovered using any suitable method, such as elutionwith a lower pH buffer, with a mild denaturing agent (e.g., urea), orwith a peptide that competes for binding to the ligand. In one example,a biotinylated target ligand is combined with a repertoire underconditions suitable for peptides or polypeptides in the repertoire tobind the target ligand (TNFR1). Bound peptides or polypeptides arerecovered using immobilized avidin or streptavidin (e.g., on a bead).

In some embodiments, the generic ligand is an antibody or antigenbinding fragment thereof. Antibodies or antigen binding fragments thatbind structural features of peptides or polypeptides that aresubstantially conserved in the peptides or polypeptides of a library orrepertoire are particularly useful as generic ligands. Antibodies andantigen binding fragments suitable for use as ligands for isolating,selecting and/or recovering protease resistant peptides or polypeptidescan be monoclonal or polyclonal and can be prepared using any suitablemethod.

Libraries/Repertoires

In other aspects, there are provided repertoires of protease resistantpeptides and polypeptides, to libraries that encode protease resistantpeptides and polypeptides, and to methods for producing such librariesand repertoires.

Libraries that encode and/or contain protease resistant peptides andpolypeptides can be prepared or obtained using any suitable method. Thelibrary can be designed to encode protease resistant peptides orpolypeptides based on a peptide or polypeptide of interest (e.g., ananti-TNFR1 peptide or polypeptide selected from a library) or can beselected from another library using the methods described herein. Forexample, a library enriched in protease resistant polypeptides can beprepared using a suitable polypeptide display system.

In one example, a phage display library comprising a repertoire ofdisplayed polypeptides comprising immunoglobulin single variable domains(e.g., V_(H), Vk, Vλ) is combined with a protease under conditionssuitable for protease activity, as described herein. Protease resistantpolypeptides are recovered based on a desired biological activity, suchas a binding activity (e.g., binding generic ligand, binding targetligand) thereby yielding a phage display library enriched in proteaseresistant polypeptides. In one embodiment, the recovery is on the basisof binding generic ligand to yield an enriched library, followed byselection of an anti-TNFR1 member of that library based on specificbinding to TNFR1.

In another example, a phage display library comprising a repertoire ofdisplayed polypeptides comprising immunoglobulin single variable domains(e.g., V_(H), Vκ, Vλ) is first screened to identify members of therepertoire that have binding specificity for a desired target antigen(TNFR1). A collection of polypeptides having the desired bindingspecificity are recovered and the collection is combined with proteaseunder conditions suitable for proteolytic activity, as described herein.A collection of protease resistant polypeptides that have the desiredtarget binding specificity is recovered, yielding a library enriched inprotease resistant and high affinity polypeptides. As described hereinin an embodiment, protease resistance in this selection methodcorrelates with high affinity binding.

Libraries that encode a repertoire of a desired type of polypeptides canreadily be produced using any suitable method. For example, a nucleicacid sequence that encodes a desired type of polypeptide (e.g., apolymerase, an immunoglobulin variable domain) can be obtained and acollection of nucleic acids that each contain one or more mutations canbe prepared, for example by amplifying the nucleic acid using anerror-prone polymerase chain reaction (PCR) system, by chemicalmutagenesis (Deng et al., J. Biol. Chem., 269:9533 (1994)) or usingbacterial mutator strains (Low et al., J. Mol. Biol., 260:359 (1996)).

In other embodiments, particular regions of the nucleic acid can betargeted for diversification. Methods for mutating selected positionsare also well known in the art and include, for example, the use ofmismatched oligonucleotides or degenerate oligonucleotides, with orwithout the use of PCR. For example, synthetic antibody libraries havebeen created by targeting mutations to the antigen binding loops. Randomor semi-random antibody H3 and L3 regions have been appended to germlineimmunoblulin V gene segments to produce large libraries with unmutatedframework regions (Hoogenboom and Winter (1992) supra; Nissim et al.(1994) supra; Griffiths et al. (1994) supra; DeKruif et al. (1995)supra). Such diversification has been extended to include some or all ofthe other antigen binding loops (Crameri et al. (1996) Nature Med.,2:100; Riechmann et al. (1995) Bio/Technology, 13:475; Morphosys, WO97/08320, supra). In other embodiments, particular regions of thenucleic acid can be targeted for diversification by, for example, atwo-step PCR strategy employing the product of the first PCR as a“mega-primer.” (See, e.g., Landt, O. et al., Gene 96:125-128 (1990).)Targeted diversification can also be accomplished, for example, by SOEPCR. (See, e.g., Horton, R. M. et al., Gene 77:61-68 (1989).)

Sequence diversity at selected positions can be achieved by altering thecoding sequence which specifies the sequence of the polypeptide suchthat a number of possible amino acids (e.g., all 20 or a subset thereof)can be incorporated at that position. Using the IUPAC nomenclature, themost versatile codon is NNK, which encodes all amino acids as well asthe TAG stop codon. The NNK codon may be used in order to introduce therequired diversity. Other codons which achieve the same ends are also ofuse, including the NNN codon, which leads to the production of theadditional stop codons TGA and TAA. Such a targeted approach can allowthe full sequence space in a target area to be explored.

The libraries can comprise protease resistant antibody polypeptides thathave a known main-chain conformation. (See, e.g., Tomlinson et al., WO99/20749.)

Libraries can be prepared in a suitable plasmid or vector. As usedherein, vector refers to a discrete element that is used to introduceheterologous DNA into cells for the expression and/or replicationthereof. Any suitable vector can be used, including plasmids (e.g.,bacterial plasmids), viral or bacteriophage vectors, artificialchromosomes and episomal vectors. Such vectors may be used for simplecloning and mutagenesis, or an expression vector can be used to driveexpression of the library. Vectors and plasmids usually contain one ormore cloning sites (e.g., a polylinker), an origin of replication and atleast one selectable marker gene. Expression vectors can further containelements to drive transcription and translation of a polypeptide, suchas an enhancer element, promoter, transcription termination signal,signal sequences, and the like. These elements can be arranged in such away as to be operably linked to a cloned insert encoding a polypeptide,such that the polypeptide is expressed and produced when such anexpression vector is maintained under conditions suitable for expression(e.g., in a suitable host cell).

Cloning and expression vectors generally contain nucleic acid sequencesthat enable the vector to replicate in one or more selected host cells.Typically in cloning vectors, this sequence is one that enables thevector to replicate independently of the host chromosomal DNA andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (e.g. SV40, adenovirus) are usefulfor cloning vectors in mammalian cells. Generally, the origin ofreplication is not needed for mammalian expression vectors, unless theseare used in mammalian cells able to replicate high levels of DNA, suchas COS cells.

Cloning or expression vectors can contain a selection gene also referredto as selectable marker. Such marker genes encode a protein necessaryfor the survival or growth of transformed host cells grown in aselective culture medium. Host cells not transformed with the vectorcontaining the selection gene will therefore not survive in the culturemedium. Typical selection genes encode proteins that confer resistanceto antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexateor tetracycline, complement auxotrophic deficiencies, or supply criticalnutrients not available in the growth media.

Suitable expression vectors can contain a number of components, forexample, an origin of replication, a selectable marker gene, one or moreexpression control elements, such as a transcription control element(e.g., promoter, enhancer, terminator) and/or one or more translationsignals, a signal sequence or leader sequence, and the like. Expressioncontrol elements and a signal or leader sequence, if present, can beprovided by the vector or other source. For example, the transcriptionaland/or translational control sequences of a cloned nucleic acid encodingan antibody chain can be used to direct expression.

A promoter can be provided for expression in a desired host cell.Promoters can be constitutive or inducible. For example, a promoter canbe operably linked to a nucleic acid encoding an antibody, antibodychain or portion thereof, such that it directs transcription of thenucleic acid. A variety of suitable promoters for procaryotic (e.g., theβ-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system, lac, tac, T3, T7 promoters for E.coli) and eucaryotic (e.g., simian virus 40 early or late promoter, Roussarcoma virus long terminal repeat promoter, cytomegalovirus promoter,adenovirus late promoter, EG-1a promoter) hosts are available.

In addition, expression vectors typically comprise a selectable markerfor selection of host cells carrying the vector, and, in the case of areplicable expression vector, an origin of replication. Genes encodingproducts which confer antibiotic or drug resistance are commonselectable markers and may be used in procaryotic (e.g., β-lactamasegene (ampicillin resistance), Tet gene for tetracycline resistance) andeucaryotic cells (e.g., neomycin (G418 or geneticin), gpt (mycophenolicacid), ampicillin, or hygromycin resistance genes). Dihydrofolatereductase marker genes permit selection with methotrexate in a varietyof hosts. Genes encoding the gene product of auxotrophic markers of thehost (e.g., LEU2, URA3, HIS3) are often used as selectable markers inyeast. Use of viral (e.g., baculovirus) or phage vectors, and vectorswhich are capable of integrating into the genome of the host cell, suchas retroviral vectors, are also contemplated.

Suitable expression vectors for expression in prokaryotic (e.g.,bacterial cells such as E. coli) or mammalian cells include, forexample, a pET vector (e.g., pET-12a, pET-36, pET-37, pET-39, pET-40,Novagen and others), a phage vector (e.g., pCANTAB 5 E, Pharmacia),pRIT2T (Protein A fusion vector, Pharmacia), pCDM8, pcDNA1.1/amp,pcDNA3.1, pRc/RSV, pEF-1 (Invitrogen, Carlsbad, Calif.), pCMV-SCRIPT,pFB, pSG5, pXT1 (Stratagene, La Jolla, Calif.), pCDEF3 (Goldman, L. A.,et al., Biotechniques, 21:1013-1015 (1996)), pSVSPORT (GibcoBRL,Rockville, Md.), pEF-Bos (Mizushima, S., et al., Nucleic Acids Res.,18:5322 (1990)) and the like. Expression vectors which are suitable foruse in various expression hosts, such as prokaryotic cells (E. coli),insect cells (Drosophila Schnieder S2 cells, Sf9), yeast (P.methanolica, P. pastoris, S. cerevisiae) and mammalian cells (e.g., COScells) are available.

Examples of vectors are expression vectors that enable the expression ofa nucleotide sequence corresponding to a polypeptide library member.Thus, selection with generic and/or target ligands can be performed byseparate propagation and expression of a single clone expressing thepolypeptide library member. As described above, the selection displaysystem may be bacteriophage display. Thus, phage or phagemid vectors maybe used. Example vectors are phagemid vectors which have an E. coli.origin of replication (for double stranded replication) and also a phageorigin of replication (for production of single-stranded DNA). Themanipulation and expression of such vectors is well known in the art(Hoogenboom and Winter (1992) supra; Nissim et al. (1994) supra).Briefly, the vector can contain a β-lactamase gene to confer selectivityon the phagemid and a lac promoter upstream of an expression cassettethat can contain a suitable leader sequence, a multiple cloning site,one or more peptide tags, one or more TAG stop codons and the phageprotein pIII. Thus, using various suppressor and non-suppressor strainsof E. coli and with the addition of glucose, iso-propylthio-β-D-galactoside (IPTG) or a helper phage, such as VCS M13, thevector is able to replicate as a plasmid with no expression, producelarge quantities of the polypeptide library member only or productphage, some of which contain at least one copy of the polypeptide-pIIIfusion on their surface.

The libraries and repertoires described herein can contain antibodyformats. For example, the polypeptide contained within the libraries andrepertoires can be whole separate V_(H) or V_(L) domains, any of whichare either modified or unmodified. scFv fragments, as well as otherantibody polypeptides, can be readily produced using any suitablemethod. A number of suitable antibody engineering methods are well knownin the art. For example, a scFv can be formed by linking nucleic acidsencoding two variable domains with a suitable oligonucleotide thatencodes an appropriate linker peptide, such as (Gly-Gly-Gly-Gly-Ser (SEQID NO: 222))₃ or other suitable linker peptides. The linker bridges theC-terminal end of the first V region and the N-terminal end of thesecond V region. Similar techniques for the construction of otherantibody formats, such as Fv, Fab and F(ab′)₂ fragments can be used. Toformat Fab and F(ab′)₂ fragments, V_(H) and V_(L) polypeptides can becombined with constant region segments, which may be isolated fromrearranged genes, germline C genes or synthesized from antibody sequencedata. A library or repertoire described herein can be a V_(H) or V_(L)library or repertoire.

The polypeptides comprising a protease resistant variable domain maycomprise a target ligand (TNFR1) binding site and a generic ligandbinding site. In certain embodiments, the generic ligand binding site isa binding site for a superantigen, such as protein A, protein L orprotein G. The variable domains can be based on any desired variabledomain, for example a human VH (e.g., V_(H) 1a, V_(H) 1b, V_(H) 2, V_(H)3, V_(H) 4, V_(H) 5, V_(H) 6), a human Vλ (e.g., VλI, VλII, VλIII, VλIV,VλV, VλVI or Vκ1) or a human Vκ (e.g., Vκ2, Vκ3, Vκ4, Vκ5, Vκ6, Vκ7,Vκ8, Vκ9 or Vκ10) or a Camelid V_(HH), optionally that has beenhumanized.

Nucleic Acids, Host Cells and Methods for Producing Protease ResistantPolypeptides

The invention relates to isolated and/or recombinant nucleic acidsencoding protease resistant peptides or polypeptides e.g., that areselectable or selected by the methods described herein.

Nucleic acids referred to herein as “isolated” are nucleic acids whichhave been separated away from other material (e.g., other nucleic acidssuch as genomic DNA, cDNA and/or RNA) in its original environment (e.g.,in cells or in a mixture of nucleic acids such as a library). Anisolated nucleic acid can be isolated as part of a vector (e.g., aplasmid).

Nucleic acids referred to herein as “recombinant” are nucleic acidswhich have been produced by recombinant DNA methodology, includingmethods which rely upon artificial recombination, such as cloning into avector or chromosome using, for example, restriction enzymes, homologousrecombination, viruses and the like, and nucleic acids prepared usingthe polymerase chain reaction (PCR).

The invention also relates to a recombinant host cell which comprises a(one or more) recombinant nucleic acid or expression constructcomprising a nucleic acid encoding a protease resistant peptide orpolypeptide, e.g., a peptide or polypeptide selectable or selected bythe methods described herein. There is also provided a method ofpreparing a protease resistant peptide or polypeptide, comprisingmaintaining a recombinant host cell of the invention under conditionsappropriate for expression of a protease resistant peptide orpolypeptide. The method can further comprise the step of isolating orrecovering the protease resistant peptide or polypeptide, if desired.

For example, a nucleic acid molecule (i.e., one or more nucleic acidmolecules) encoding a protease resistant peptide or polypeptide, or anexpression construct (i.e., one or more constructs) comprising suchnucleic acid molecule(s), can be introduced into a suitable host cell tocreate a recombinant host cell using any method appropriate to the hostcell selected (e.g., transformation, transfection, electroporation,infection), such that the nucleic acid molecule(s) are operably linkedto one or more expression control elements (e.g., in a vector, in aconstruct created by processes in the cell, integrated into the hostcell genome). The resulting recombinant host cell can be maintainedunder conditions suitable for expression (e.g., in the presence of aninducer, in a suitable animal, in suitable culture media supplementedwith appropriate salts, growth factors, antibiotics, nutritionalsupplements, etc.), whereby the encoded peptide or polypeptide isproduced. If desired, the encoded peptide or polypeptide can be isolatedor recovered (e.g., from the animal, the host cell, medium, milk). Thisprocess encompasses expression in a host cell of a transgenic animal(see, e.g., WO 92/03918, GenPharm International).

The protease resistant peptide or polypeptide selected by the methoddescribed herein can also be produced in a suitable in vitro expressionsystem, by chemical synthesis or by any other suitable method. Thus, thepresent invention provides for protease resistant peptides andpolypeptides.

Polypeptides, DAB™s & Antagonists

As described and exemplified herein, protease resistant DAB™s of theinvention generally bind their target ligand with high affinity. Thus,in another aspect, there is provided a method for selecting, isolatingand/or recovering a polypeptide or DAB™ of the invention that bindsTNFR1 with high affinity. Generally, the method comprises providing alibrary or repertoire of peptides or polypeptides (e.g. DAB™s),combining the library or repertoire with a protease (e.g., trypsin,elastase, leucozyme, pancreatin, sputum) under conditions suitable forprotease activity, and selecting, isolating and/or recovering a peptideor polypeptide that binds a ligand (e.g., target ligand). Because thelibrary or repertoire has been exposed to protease under conditionswhere protease sensitive peptides or polypeptides will be digested, theactivity of protease can eliminate the less stable polypeptides thathave low binding affinity, and thereby produce a collection of highaffinity binding peptides or polypeptides. For example, the polypeptideor DAB™ of the invention can bind TNFR1 with an affinity (K_(D);K_(D)=K_(off)(kd)/K_(on)(ka) as determined by surface plasmon resonance)of 1 μM or stronger, or about 500 nM to about 0.5 pM. For example, thepolypeptide or DAB™ of the invention can bind TNFR1 with an affinity ofabout 500 nM, about 100 nM, about 10 nM, about 1 nM, about 500 pM, about100 pM, about 10 pM, about 1 pM or about 0.5 pM. Although we are notbound by any particular theory, peptides and polypeptides that areresistant to proteases are believed to have a lower entropy and/or ahigher stabilization energy. Thus, the correlation between proteaseresistance and high affinity binding may be related to the compactnessand stability of the surfaces of the peptides and polypeptides and DAB™sselected by the method described herein.

In one embodiment, the polypeptide, DAB™ or antagonist of the inventioninhibits binding of TNF alpha to TNF alpha Receptor I (p55 receptor)with an inhibitory concentration 50 (IC50) of or about 500 nM to 50 pM,or 100 nM to 50 pM, or 10 nM to 100 pM, or 1 nM to 100 pM; for example50 nM or less, or 5 nM or less, or 500 pM or less, or 200 pM or less, or100 pM or less.

In certain embodiments, the polypeptide, DAB™ or antagonist specificallybinds TNFR1, e.g., human TNFRI, and dissociates from human TNFR1 with adissociation constant (K_(D)) of 300 nM to 1 pM or 300 nM to 5 pM or 50nM to 1 pM or 50 nM to 5 pM or 50 nM to 20 pM or about 10 pM or about 15pM or about 20 pM as determined by surface plasmon resonance. In certainembodiments, the polypeptide, DAB™ or antagonist specifically bindsTNFR1, e.g., human TNFRI, and dissociates from human TNFR1 with aK_(off) rate constant of 5×10⁻¹ s⁻¹ to 1×10⁻⁷ s⁻¹ or 1×10⁻³ s⁻¹ to1×10⁻⁷ s⁻¹ or 1×10⁻⁴ s⁻¹ to 1×10⁻⁷ s⁻¹ or 1×10⁻⁵ s⁻¹ to 1×10⁻⁷ s⁻¹ or1×10⁻⁴ s⁻¹ or 1×10⁻⁵ s⁻¹ as determined by surface plasmon resonance. Incertain embodiments, the polypeptide, DAB™ or antagonist specificallybinds TNFR1, e.g., human TNFRI, with a K_(on) of 1×10⁻³ M⁻¹s⁻¹ to 1×10⁻⁷M⁻¹s⁻¹ or 1×10⁻³ M⁻¹s⁻¹ to 1×10⁻⁶ M⁻¹s⁻¹ or about 1×10⁻⁴ M⁻¹s⁻¹ or about1×10⁻⁵ M⁻¹s⁻¹. In one embodiment, the polypeptide, DAB™ or antagonistspecifically binds TNFR1, e.g., human TNFRI, and dissociates from humanTNFR1 with a dissociation constant (K_(D)) and a K_(off) as defined inthis paragraph. In one embodiment, the polypeptide, DAB™ or antagonistspecifically binds TNFR1, e.g., human TNFRI, and dissociates from humanTNFR1 with a dissociation constant (K_(D)) and a K_(on) as defined inthis paragraph. In some embodiments, the polypeptide or DAB™specifically binds TNFR1 (e.g., human TNFR1) with a K_(D) and/or K_(off)and/or K_(on) as recited in this paragraph and comprises an amino acidsequence that is at least or at least about 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence of DOM1h-131-206 (shown in FIG. 3).

The polypeptide, DAB™ or antagonist can be expressed in E. coli or inPichia species (e.g., P. pastoris). In one embodiment, the ligand orDAB™ monomer is secreted in a quantity of at least about 0.5 mg/L whenexpressed in E. coli or in Pichia species (e.g., P. pastoris). Although,the ligands and DAB™ monomers described herein can be secretable whenexpressed in E. coli or in Pichia species (e.g., P. pastoris), they canbe produced using any suitable method, such as synthetic chemicalmethods or biological production methods that do not employ E. coli orPichia species.

In some embodiments, the polypeptide, DAB™ or antagonist does notcomprise a Camelid immunoglobulin variable domain, or one or moreframework amino acids that are unique to immunoglobulin variable domainsencoded by Camelid germline antibody gene segments, e.g. at position108, 37, 44, 45 and/or 47.

Antagonists of TNFR1 according to the invention can be monovalent ormultivalent. In some embodiments, the antagonist is monovalent andcontains one binding site that interacts with TNFR1, the binding siteprovided by a polypeptide or DAB™ of the invention. Monovalentantagonists bind one TNFR1 and may not induce cross-linking orclustering of TNFR1 on the surface of cells which can lead to activationof the receptor and signal transduction.

In other embodiments, the antagonist of TNFR1 is multivalent.Multivalent antagonists of TNFR1 can contain two or more copies of aparticular binding site for TNFR1 or contain two or more differentbinding sites that bind TNFR1, at least one of the binding sites beingprovided by a polypeptide or DAB™ of the invention. For example, asdescribed herein the antagonist of TNFR1 can be a dimer, trimer ormultimer comprising two or more copies of a particular polypeptide orDAB™ of the invention that binds TNFR1, or two or more differentpolypeptides or DAB™s of the invention that bind TNFR1. In oneembodiment, a multivalent antagonist of TNFR1 does not substantiallyagonize TNFR1 (act as an agonist of TNFR1) in a standard cell assay(i.e., when present at a concentration of 1 nM, 10 nM, 100 nM, 1 μM, 10μM, 100 μM, 1000 μM or 5,000 μM, results in no more than about 5% of theTNFR1-mediated activity induced by TNFα (100 pg/ml) in the assay).

In certain embodiments, the multivalent antagonist of TNFR1 contains twoor more binding sites for a desired epitope or domain of TNFR1. Forexample, the multivalent antagonist of TNFR1 can comprise two or morebinding sites that bind the same epitope in Domain 1 of TNFR1.

In other embodiments, the multivalent antagonist of TNFR1 contains twoor more binding sites provided by polypeptides or DAB™s of the inventionthat bind to different epitopes or domains of TNFR1. In one embodiment,such multivalent antagonists do not agonize TNFR1 when present at aconcentration of about 1 nM, or about 10 nM, or about 100 nM, or about 1μM, or about 10 μM, in a standard L929 cytotoxicity assay or a standardHeLa IL-8 assay as described in WO2006038027.

Other antagonists of TNFR1 do no inhibit binding of TNFα to TNFR1. Suchligands (and antagonists) may have utility as diagnostic agents, becausethey can be used to bind and detect, quantify or measure TNFR1 in asample and will not compete with TNF in the sample for binding to TNFR1.Accordingly, an accurate determination of whether or how much TNFR1 isin the sample can be made.

In other embodiments, the polypeptide, DAB™ or antagonist specificallybinds TNFR1 with a K_(D) described herein and inhibits lethality in astandard mouse LPS/D-galactosamine-induced septic shock model (i.e.,prevents lethality or reduces lethality by at least about 10%, ascompared with a suitable control). In one embodiment, the polypeptide,DAB™ or antagonist inhibits lethality by at least about 25%, or by atleast about 50%, as compared to a suitable control in a standard mouseLPS/D-galactosamine-induced septic shock model when administered atabout 5 mg/kg or more, for example about 1 mg/kg.

In other embodiments, the polypeptide, DAB™ or antagonist binds TNFRIand antagonizes the activity of the TNFR1 in a standard cell assay withan ND₅₀ of ≦100 nM, and at a concentration of ≦10 μM the DAB™ agonizesthe activity of the TNFR1 by ≦5% in the assay.

In particular embodiments, the polypeptide, DAB™ or antagonist does notsubstantially agonize TNFR1 (act as an agonist of TNFR1) in a standardcell assay (i.e., when present at a concentration of 1 nM, 10 nM, 100nM, 1 μM, 10 μM, 100 μM, 1000 μM or 5,000 μM, results in no more thanabout 5% of the TNFR1-mediated activity induced by TNFα (100 pg/ml) inthe assay).

In certain embodiments, the polypeptide, DAB™ or antagonist of theinvention are efficacious in models of chronic inflammatory diseaseswhen an effective amount is administered. Generally an effective amountis about 1 mg/kg to about 10 mg/kg (e.g., about 1 mg/kg, about 2 mg/kg,about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7mg/kg, about 8 mg/kg, about 9 mg/kg, or about 10 mg/kg). The models ofchronic inflammatory disease (see those described in WO2006038027) arerecognized by those skilled in the art as being predictive oftherapeutic efficacy in humans.

In particular embodiments, the polypeptide, DAB™ or antagonist isefficacious in the standard mouse collagen-induced arthritis model (seeWO2006038027 for details of the model). For example, administering aneffective amount of the polypeptide, DAB™ or antagonist can reduce theaverage arthritic score of the summation of the four limbs in thestandard mouse collagen-induced arthritis model, for example, by about 1to about 16, about 3 to about 16, about 6 to about 16, about 9 to about16, or about 12 to about 16, as compared to a suitable control. Inanother example, administering an effective amount of the polypeptide,DAB™ or antagonist can delay the onset of symptoms of arthritis in thestandard mouse collagen-induced arthritis model, for example, by about 1day, about 2 days, about 3 days, about 4 days, about 5 days, about 6days, about 7 days, about 10 days, about 14 days, about 21 days or about28 days, as compared to a suitable control. In another example,administering an effective amount of the polypeptide, DAB™ or antagonistcan result in an average arthritic score of the summation of the fourlimbs in the standard mouse collagen-induced arthritis model of 0 toabout 3, about 3 to about 5, about 5 to about 7, about 7 to about 15,about 9 to about 15, about 10 to about 15, about 12 to about 15, orabout 14 to about 15.

In other embodiments, the polypeptide, DAB™ or antagonist is efficaciousin the mouse ΔARE model of arthritis (see WO2006038027 for details ofthe model). For example, administering an effective amount of thepolypeptide, DAB™ or antagonist can reduce the average arthritic scorein the mouse ΔARE model of arthritis, for example, by about 0.1 to about2.5, about 0.5 to about 2.5, about 1 to about 2.5, about 1.5 to about2.5, or about 2 to about 2.5, as compared to a suitable control. Inanother example, administering an effective amount of the polypeptide,DAB™ or antagonist can delay the onset of symptoms of arthritis in themouse ΔARE model of arthritis by, for example, about 1 day, about 2days, about 3 days, about 4 days, about 5 days, about 6 days, about 7days, about 10 days, about 14 days, about 21 days or about 28 days, ascompared to a suitable control. In another example, administering aneffective amount of the polypeptide, DAB™ or antagonist can result in anaverage arthritic score in the mouse ΔARE model of arthritis of 0 toabout 0.5, about 0.5 to about 1, about 1 to about 1.5, about 1.5 toabout 2, or about 2 to about 2.5.

In other embodiments, the polypeptide, DAB™ or antagonist is efficaciousin the mouse ΔARE model of inflammatory bowel disease (IBD) (seeWO2006038027 for details of the model). For example, administering aneffective amount of the polypeptide, DAB™ or antagonist can reduce theaverage acute and/or chronic inflammation score in the mouse ΔARE modelof IBD, for example, by about 0.1 to about 2.5, about 0.5 to about 2.5,about 1 to about 2.5, about 1.5 to about 2.5, or about 2 to about 2.5,as compared to a suitable control. In another example, administering aneffective amount of the polypeptide, DAB™ or antagonist can delay theonset of symptoms of IBD in the mouse ΔARE model of IBD by, for example,about 1 day, about 2 days, about 3 days, about 4 days, about 5 days,about 6 days, about 7 days, about 10 days, about 14 days, about 21 daysor about 28 days, as compared to a suitable control. In another example,administering an effective amount of the polypeptide, DAB™ or antagonistcan result in an average acute and/or chronic inflammation score in themouse ΔARE model of IBD of 0 to about 0.5, about 0.5 to about 1, about 1to about 1.5, about 1.5 to about 2, or about 2 to about 2.5.

In other embodiments, the polypeptide, DAB™ or antagonist is efficaciousin the mouse dextran sulfate sodium (DSS) induced model of IBD (seeWO2006038027 for details of the model). For example, administering aneffective amount of the polypeptide, DAB™ or antagonist can reduce theaverage severity score in the mouse DSS model of IBD, for example, byabout 0.1 to about 2.5, about 0.5 to about 2.5, about 1 to about 2.5,about 1.5 to about 2.5, or about 2 to about 2.5, as compared to asuitable control. In another example, administering an effective amountof the polypeptide, DAB™ or antagonist can delay the onset of symptomsof IBD in the mouse DSS model of IBD by, for example, about 1 day, about2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7days, about 10 days, about 14 days, about 21 days or about 28 days, ascompared to a suitable control. In another example, administering aneffective amount of the polypeptide, DAB™ or antagonist can result in anaverage severity score in the mouse DSS model of IBD of 0 to about 0.5,about 0.5 to about 1, about 1 to about 1.5, about 1.5 to about 2, orabout 2 to about 2.5.

In particular embodiments, the polypeptide, DAB™ or antagonist isefficacious in the mouse tobacco smoke model of chronic obstructivepulmonary disease (COPD) (see WO2006038027 and WO2007049017 for detailsof the model). For example, administering an effective amount of theligand can reduce or delay onset of the symptoms of COPD, as compared toa suitable control.

Animal model systems which can be used to screen the effectiveness ofthe antagonists of TNFR1 (e.g., ligands, antibodies or binding proteinsthereof) in protecting against or treating the disease are available.Methods for the testing of systemic lupus erythematosus (SLE) insusceptible mice are known in the art (Knight et al. (1978) J. Exp.Med., 147: 1653; Reinersten et al. (1978) New Eng. J. Med., 299: 515).Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing thedisease with soluble AchR protein from another species (Lindstrom et al.(1988) Adv. Immunol., 42: 233). Arthritis is induced in a susceptiblestrain of mice by injection of Type II collagen (Stuart et al. (1984)Ann. Rev. Immunol., 42: 233). A model by which adjuvant arthritis isinduced in susceptible rats by injection of mycobacterial heat shockprotein has been described (Van Eden et al. (1988) Nature, 331: 171).Thyroiditis is induced in mice by administration of thyroglobulin asdescribed (Maron et al. (1980) J. Exp. Med., 152: 1115). Insulindependent diabetes mellitus (IDDM) occurs naturally or can be induced incertain strains of mice such as those described by Kanasawa et al.(1984) Diabetologia, 27: 113. EAE in mouse and rat serves as a model forMS in human. In this model, the demyelinating disease is induced byadministration of myelin basic protein (see Paterson (1986) Textbook ofImmunopathology, Mischer et al., eds., Grune and Stratton, New York, pp.179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al.(1987) J. Immunol., 138: 179).

Generally, the present ligands (e.g., antagonists) will be utilised inpurified form together with pharmacologically appropriate carriers.Typically, these carriers include aqueous or alcoholic/aqueoussolutions, emulsions or suspensions, any including saline and/orbuffered media. Parenteral vehicles include sodium chloride solution,Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.Suitable physiologically-acceptable adjuvants, if necessary to keep apolypeptide complex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition). A variety ofsuitable formulations can be used, including extended releaseformulations.

The ligands (e.g., antagonists) of the present invention may be used asseparately administered compositions or in conjunction with otheragents. These can include various immunotherapeutic drugs, such ascylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins.Pharmaceutical compositions can include “cocktails” of various cytotoxicor other agents in conjunction with the ligands of the presentinvention, or even combinations of ligands according to the presentinvention having different specificities, such as ligands selected usingdifferent target antigens or epitopes, whether or not they are pooledprior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected ligands thereof of the invention can beadministered to any patient in accordance with standard techniques.

The administration can be by any appropriate mode, includingparenterally, intravenously, intramuscularly, intraperitoneally,transdermally, via the pulmonary route, or also, appropriately, bydirect infusion with a catheter. The dosage and frequency ofadministration will depend on the age, sex and condition of the patient,concurrent administration of other drugs, counterindications and otherparameters to be taken into account by the clinician. Administration canbe local (e.g., local delivery to the lung by pulmonary administration,e.g., intranasal administration) or systemic as indicated.

The ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilisation and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilisationand reconstitution can lead to varying degrees of antibody activity loss(e.g. with conventional immunoglobulins, IgM antibodies tend to havegreater activity loss than IgG antibodies) and that use levels may haveto be adjusted upward to compensate.

The compositions containing the present ligands (e.g., antagonists) or acocktail thereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of ligand, e.g. DAB™ or antagonist perkilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being morecommonly used. For prophylactic applications, compositions containingthe present ligands or cocktails thereof may also be administered insimilar or slightly lower dosages, to prevent, inhibit or delay onset ofdisease (e.g., to sustain remission or quiescence, or to prevent acutephase). The skilled clinician will be able to determine the appropriatedosing interval to treat, suppress or prevent disease. When an ligand ofTNFR1 (e.g., antagonist) is administered to treat, suppress or prevent achronic inflammatory disease, it can be administered up to four timesper day, twice weekly, once weekly, once every two weeks, once a month,or once every two months, at a dose off, for example, about 10 μg/kg toabout 80 mg/kg, about 100 μg/kg to about 80 mg/kg, about 1 mg/kg toabout 80 mg/kg, about 1 mg/kg to about 70 mg/kg, about 1 mg/kg to about60 mg/kg, about 1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 40mg/kg, about 1 mg/kg to about 30 mg/kg, about 1 mg/kg to about 20 mg/kg,about 1 mg/kg to about 10 mg/kg, about 10 μg/kg to about 10 mg/kg, about10 μg/kg to about 5 mg/kg, about 10 μg/kg to about 2.5 mg/kg, about 1mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg.In particular embodiments, the ligand of TNFR1 (e.g., antagonist) isadministered to treat, suppress or prevent a chronic inflammatorydisease once every two weeks or once a month at a dose of about 10 μg/kgto about 10 mg/kg (e.g., about 10 μg/kg, about 100 μg/kg, about 1 mg/kg,about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg or about 10 mg/kg.)

Treatment or therapy performed using the compositions described hereinis considered “effective” if one or more symptoms are reduced (e.g., byat least 10% or at least one point on a clinical assessment scale),relative to such symptoms present before treatment, or relative to suchsymptoms in an individual (human or model animal) not treated with suchcomposition or other suitable control. Symptoms will obviously varydepending upon the disease or disorder targeted, but can be measured byan ordinarily skilled clinician or technician. Such symptoms can bemeasured, for example, by monitoring the level of one or morebiochemical indicators of the disease or disorder (e.g., levels of anenzyme or metabolite correlated with the disease, affected cell numbers,etc.), by monitoring physical manifestations (e.g., inflammation, tumorsize, etc.), or by an accepted clinical assessment scale, for example,the Expanded Disability Status Scale (for multiple sclerosis), theIrvine Inflammatory Bowel Disease Questionnaire (32 point assessmentevaluates quality of life with respect to bowel function, systemicsymptoms, social function and emotional status—score ranges from 32 to224, with higher scores indicating a better quality of life), theQuality of Life Rheumatoid Arthritis Scale, or other accepted clinicalassessment scale as known in the field. A sustained (e.g., one day ormore, or longer) reduction in disease or disorder symptoms by at least10% or by one or more points on a given clinical scale is indicative of“effective” treatment. Similarly, prophylaxis performed using acomposition as described herein is “effective” if the onset or severityof one or more symptoms is delayed, reduced or abolished relative tosuch symptoms in a similar individual (human or animal model) nottreated with the composition.

A composition containing a ligand (e.g., antagonist) or cocktail thereofaccording to the present invention may be utilised in prophylactic andtherapeutic settings to aid in the alteration, inactivation, killing orremoval of a select target cell population in a mammal. In addition, theselected repertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the ligands whereby the undesired cells are killedor otherwise removed from the blood for return to the mammal inaccordance with standard techniques.

A composition containing a ligand (e.g., antagonist) according to thepresent invention may be utilised in prophylactic and therapeuticsettings to aid in the alteration, inactivation, killing or removal of aselect target cell population in a mammal.

The ligands (e.g., anti-TNFR1 antagonists, DAB™ monomers) can beadministered and or formulated together with one or more additionaltherapeutic or active agents. When a ligand (e.g., a DAB™) isadministered with an additional therapeutic agent, the ligand can beadministered before, simultaneously with or subsequent to administrationof the additional agent. Generally, the ligand and additional agent areadministered in a manner that provides an overlap of therapeutic effect.

In one embodiment, the invention is a method for treating, suppressingor preventing a chronic inflammatory disease, comprising administeringto a mammal in need thereof a therapeutically-effective dose or amountof a polypeptide, DAB™ or antagonist of TNFR1 according to theinvention.

In one embodiment, the invention is a method for treating, suppressingor preventing arthritis (e.g., rheumatoid arthritis, juvenile rheumatoidarthritis, ankylosing spondylitis, psoriatic arthritis) comprisingadministering to a mammal in need thereof a therapeutically-effectivedose or amount of a polypeptide, DAB™ or antagonist of TNFR1 accordingto the invention.

In another embodiment, the invention is a method for treating,suppressing or preventing psoriasis comprising administering to a mammalin need thereof a therapeutically-effective dose or amount of apolypeptide, DAB™ or antagonist of TNFR1 according to the invention.

In another embodiment, the invention is a method for treating,suppressing or preventing inflammatory bowel disease (e.g., Crohn'sdisease, ulcerative colitis) comprising administering to a mammal inneed thereof a therapeutically-effective dose or amount of apolypeptide, DAB™ or antagonist of TNFR1 according to the invention.

In another embodiment, the invention is a method for treating,suppressing or preventing chronic obstructive pulmonary disease (e.g.,chronic bronchitis, chronic obstructive bronchitis, emphysema),comprising administering to a mammal in need thereof atherapeutically-effective dose or amount of a polypeptide, DAB™ orantagonist of TNFR1 according to the invention.

In another embodiment, the invention is a method for treating,suppressing or preventing pneumonia (e.g., bacterial pneumonia, such asStaphylococcal pneumonia) comprising administering to a mammal in needthereof a therapeutically-effective dose or amount of a polypeptide,DAB™ or antagonist of TNFR1 according to the invention.

The invention provides a method for treating, suppressing or preventingother pulmonary diseases in addition to chronic obstructive pulmonarydisease, and pneumonia. Other pulmonary diseases that can be treated,suppressed or prevented in accordance with the invention include, forexample, cystic fibrosis and asthma (e.g., steroid resistant asthma).Thus, in another embodiment, the invention is a method for treating,suppressing or preventing a pulmonary disease (e.g., cystic fibrosis,asthma) comprising administering to a mammal in need thereof atherapeutically-effective dose or amount of a polypeptide, DAB™ orantagonist of TNFR1 according to the invention.

In particular embodiments, an antagonist of TNFR1 is administered viapulmonary delivery, such as by inhalation (e.g., intrabronchial,intranasal or oral inhalation, intranasal drops) or by systemic delivery(e.g., parenteral, intravenous, intramuscular, intraperitoneal,subcutaneous).

In another embodiment, the invention is a method treating, suppressingor preventing septic shock comprising administering to a mammal in needthereof a therapeutically-effective dose or amount of a polypeptide,DAB™ or antagonist of TNFR1 according to the invention.

In a further aspect of the invention, there is provided a compositioncomprising a a polypeptide, DAB™ or antagonist of TNFR1 according to theinvention and a pharmaceutically acceptable carrier, diluent orexcipient.

Moreover, the present invention provides a method for the treatment ofdisease using a polypeptide, DAB™ or antagonist of TNFR1 or acomposition according to the present invention. In an embodiment thedisease is cancer or an inflammatory disease, e.g. rheumatoid arthritis,asthma or Crohn's disease.

Formats

Increased half-life is useful in in vivo applications ofimmunoglobulins, especially antibodies and most especially antibodyfragments of small size. Such fragments (Fvs, disulphide bonded Fvs,Fabs, scFvs, DAB™s) suffer from rapid clearance from the body; thus,whilst they are able to reach most parts of the body rapidly, and arequick to produce and easier to handle, their in vivo applications havebeen limited by their only brief persistence in vivo. One embodiment ofthe invention solves this problem by providing increased half-life ofthe ligands in vivo and consequently longer persistence times in thebody of the functional activity of the ligand.

Methods for pharmacokinetic analysis and determination of ligandhalf-life will be familiar to those skilled in the art. Details may befound in Kenneth, A et al: Chemical Stability of Pharmaceuticals: AHandbook for Pharmacists and in Peters et al, pharmacokinetic analysis:A Practical Approach (1996). Reference is also made to“Pharmacokinetics”, M Gibaldi & D Perron, published by Marcel Dekker,2^(nd) Rev. ex edition (1982), which describes pharmacokineticparameters such as t alpha and t beta half lives and area under thecurve (AUC).

Half lives (t½ alpha and t½ beta) and AUC can be determined from a curveof serum concentration of ligand against time. The WINNONLIN™ analysispackage (available from Pharsight Corp., Mountain View, Calif. 94040,USA) can be used, for example, to model the curve. In a first phase (thealpha phase) the ligand is undergoing mainly distribution in thepatient, with some elimination. A second phase (beta phase) is theterminal phase when the ligand has been distributed and the serumconcentration is decreasing as the ligand is cleared from the patient.The t alpha half life is the half life of the first phase and the t betahalf life is the half life of the second phase. Thus, in one embodiment,the present invention provides a ligand or a composition comprising aligand according to the invention having a tα half-life in the range of15 minutes or more. In one embodiment, the lower end of the range is 30minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition, oralternatively, a ligand or composition according to the invention willhave a tα half life in the range of up to and including 12 hours. In oneembodiment, the upper end of the range is 11, 10, 9, 8, 7, 6 or 5 hours.An example of a suitable range is 1 to 6 hours, 2 to 5 hours or 3 to 4hours.

In one embodiment, the present invention provides a ligand (polypeptide,DAB™ or antagonist) or a composition comprising a ligand according tothe invention having a tβ half-life in the range of 2.5 hours or more.In one embodiment, the lower end of the range is 3 hours, 4 hours, 5hours, 6 hours, 7 hours, 10 hours, 11 hours, or 12 hours. In addition,or alternatively, a ligand or composition according to the invention hasa tβ half-life in the range of up to and including 21 days. In oneembodiment, the upper end of the range is 12 hours, 24 hours, 2 days, 3days, 5 days, 10 days, 15 days or 20 days. In one embodiment a ligand orcomposition according to the invention will have a tβ half life in therange 12 to 60 hours. In a further embodiment, it will be in the range12 to 48 hours. In a further embodiment still, it will be in the range12 to 26 hours.

In addition, or alternatively to the above criteria, the presentinvention provides a ligand or a composition comprising a ligandaccording to the invention having an AUC value (area under the curve) inthe range of 1 mg·min/ml or more. In one embodiment, the lower end ofthe range is 5, 10, 15, 20, 30, 100, 200 or 300 mg·min/ml. In addition,or alternatively, a ligand or composition according to the invention hasan AUC in the range of up to 600 mg·min/ml. In one embodiment, the upperend of the range is 500, 400, 300, 200, 150, 100, 75 or 50 mg·min/ml. Inone embodiment a ligand according to the invention will have a AUC inthe range selected from the group consisting of the following: 15 to 150mg·min/ml, 15 to 100 mg·min/ml, 15 to 75 mg·min/ml, and 15 to 50mg·min/ml.

Polypeptides and DAB™s of the invention and antagonists comprising thesecan be formatted to have a larger hydrodynamic size, for example, byattachment of a PEG group, serum albumin, transferrin, transferrinreceptor or at least the transferrin-binding portion thereof, anantibody Fc region, or by conjugation to an antibody domain. Forexample, polypeptides DAB™s and antagonists formatted as a largerantigen-binding fragment of an antibody or as an antibody (e.g.,formatted as a Fab, Fab′, F(ab)₂, F(ab′)₂, IgG, scFv).

Hydrodynamic size of the ligands (e.g., DAB™ monomers and multimers) ofthe invention may be determined using methods which are well known inthe art. For example, gel filtration chromatography may be used todetermine the hydrodynamic size of a ligand. Suitable gel filtrationmatrices for determining the hydrodynamic sizes of ligands, such ascross-linked agarose matrices, are well known and readily available.

The size of a ligand format (e.g., the size of a PEG moiety attached toa DAB™ monomer), can be varied depending on the desired application. Forexample, where ligand is intended to leave the circulation and enterinto peripheral tissues, it is desirable to keep the hydrodynamic sizeof the ligand low to facilitate extravazation from the blood stream.Alternatively, where it is desired to have the ligand remain in thesystemic circulation for a longer period of time the size of the ligandcan be increased, for example by formatting as an Ig like protein.

Half-Life Extension by Targeting an Antigen or Epitope that IncreasesHalf-Live In Vivo

The hydrodynamic size of a ligand and its serum half-life can also beincreased by conjugating or associating a TNFR1 binding polypeptide,DAB™ or antagonist of the invention to a binding domain (e.g., antibodyor antibody fragment) that binds an antigen or epitope that increaseshalf-live in vivo, as described herein. For example, the TNFR1 bindingagent (e.g., polypeptide) can be conjugated or linked to an anti-serumalbumin or anti-neonatal Fc receptor antibody or antibody fragment, e.g.an anti-SA or anti-neonatal Fc receptor DAB™, Fab, Fab′ or scFv, or toan anti-SA affibody or anti-neonatal Fc receptor Affibody or an anti-SAavimer, or an anti-SA binding domain which comprises a scaffold selectedfrom, but preferably not limited to, the group consisting of CTLA-4,lipocallin, SpA, an affibody, an avimer, GroEl and fibronectin (seePCT/GB2008/000453 filed 8 Feb. 2008 for disclosure of these bindingdomain, which domains and their sequences are incorporated herein byreference and form part of the disclosure of the present text).Conjugating refers to a composition comprising polypeptide, DAB™ orantagonist of the invention that is bonded (covalently or noncovalently)to a binding domain that binds serum albumin.

Suitable polypeptides that enhance serum half-life in vivo include, forexample, transferrin receptor specific ligand-neuropharmaceutical agentfusion proteins (see U.S. Pat. No. 5,977,307, the teachings of which areincorporated herein by reference), brain capillary endothelial cellreceptor, transferrin, transferrin receptor (e.g., soluble transferrinreceptor), insulin, insulin-like growth factor 1 (IGF 1) receptor,insulin-like growth factor 2 (IGF 2) receptor, insulin receptor, bloodcoagulation factor X, al-antitrypsin and HNF 1α. Suitable polypeptidesthat enhance serum half-life also include alpha-1 glycoprotein(orosomucoid; AAG), alpha-1 antichymotrypsin (ACT), alpha-1microglobulin (protein HC; AIM), antithrombin III (AT III),apolipoprotein A-1 (Apo A-1), apolipoprotein B (Apo B), ceruloplasmin(Cp), complement component C3 (C3), complement component C4 (C4), C1esterase inhibitor (C1 INH), C-reactive protein (CRP), ferritin (FER),hemopexin (HPX), lipoprotein(a) (Lp(a)), mannose-binding protein (MBP),myoglobin (Myo), prealbumin (transthyretin; PAL), retinol-bindingprotein (RBP), and rheumatoid factor (RF).

Suitable proteins from the extracellular matrix include, for example,collagens, laminins, integrins and fibronectin. Collagens are the majorproteins of the extracellular matrix. About 15 types of collagenmolecules are currently known, found in different parts of the body,e.g. type I collagen (accounting for 90% of body collagen) found inbone, skin, tendon, ligaments, cornea, internal organs or type IIcollagen found in cartilage, vertebral disc, notochord, and vitreoushumor of the eye.

Suitable proteins from the blood include, for example, plasma proteins(e.g., fibrin, α-2 macroglobulin, serum albumin, fibrinogen (e.g.,fibrinogen A, fibrinogen B), serum amyloid protein A, haptoglobin,profilin, ubiquitin, uteroglobulin and β-2-microglobulin), enzymes andenzyme inhibitors (e.g., plasminogen, lysozyme, cystatin C,alpha-1-antitrypsin and pancreatic trypsin inhibitor), proteins of theimmune system, such as immunoglobulin proteins (e.g., IgA, IgD, IgE,IgG, IgM, immunoglobulin light chains (kappa/lambda)), transportproteins (e.g., retinol binding protein, α-1 microglobulin), defensins(e.g., beta-defensin 1, neutrophil defensin 1, neutrophil defensin 2 andneutrophil defensin 3) and the like.

Suitable proteins found at the blood brain barrier or in neural tissueinclude, for example, melanocortin receptor, myelin, ascorbatetransporter and the like.

Suitable polypeptides that enhance serum half-life in vivo also includeproteins localized to the kidney (e.g., polycystin, type IV collagen,organic anion transporter K1, Heymann's antigen), proteins localized tothe liver (e.g., alcohol dehydrogenase, G250), proteins localized to thelung (e.g., secretory component, which binds IgA), proteins localized tothe heart (e.g., HSP 27, which is associated with dilatedcardiomyopathy), proteins localized to the skin (e.g., keratin), bonespecific proteins such as morphogenic proteins (BMPs), which are asubset of the transforming growth factor β superfamily of proteins thatdemonstrate osteogenic activity (e.g., BMP-2, BMP-4, BMP-5, BMP-6,BMP-7, BMP-8), tumor specific proteins (e.g., trophoblast antigen,herceptin receptor, oestrogen receptor, cathepsins (e.g., cathepsin B,which can be found in liver and spleen)).

Suitable disease-specific proteins include, for example, antigensexpressed only on activated T-cells, including LAG-3 (lymphocyteactivation gene), osteoprotegerin ligand (OPGL; see Nature 402, 304-309(1999)), OX40 (a member of the TNF receptor family, expressed onactivated T cells and specifically up-regulated in human T cell leukemiavirus type-I (HTLV-I)-producing cells; see Immunol. 165 (1):263-70(2000)). Suitable disease-specific proteins also include, for example,metalloproteases (associated with arthritis/cancers) including CG6512Drosophila, human paraplegin, human FtsH, human AFG3L2, murine ftsH; andangiogenic growth factors, including acidic fibroblast growth factor(FGF-1), basic fibroblast growth factor (FGF-2), vascular endothelialgrowth factor/vascular permeability factor (VEGF/VPF), transforminggrowth factor-α (TGF α), tumor necrosis factor-alpha (TNF-α),angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet-derivedendothelial growth factor (PD-ECGF), placental growth factor (P1GF),midkine platelet-derived growth factor-BB (PDGF), and fractalkine.

Suitable polypeptides that enhance serum half-life in vivo also includestress proteins such as heat shock proteins (HSPs). HSPs are normallyfound intracellularly. When they are found extracellularly, it is anindicator that a cell has died and spilled out its contents. Thisunprogrammed cell death (necrosis) occurs when as a result of trauma,disease or injury, extracellular HSPs trigger a response from the immunesystem. Binding to extracellular HSP can result in localizing thecompositions of the invention to a disease site.

Suitable proteins involved in Fc transport include, for example,Brambell receptor (also known as FcRB). This Fc receptor has twofunctions, both of which are potentially useful for delivery. Thefunctions are (1) transport of IgG from mother to child across theplacenta (2) protection of IgG from degradation thereby prolonging itsserum half-life. It is thought that the receptor recycles IgG fromendosomes. (See, Holliger et al, Nat Biotechnol 15(7):632-6 (1997).)

DAB™s that Bind Serum Albumin

The invention in one embodiment provides a polypeptide or antagonist(e.g., dual specific ligand comprising an anti-TNFR1 DAB™ (a firstDAB™)) that binds to TNFR1 and a second DAB™ that binds serum albumin(SA), the second DAB™ binding SA with a K_(D) as determined by surfaceplasmon resonance of 1 nM to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70,100, 200, 300, 400 or 500 μM (i.e., ×10⁻⁹ to 5×10⁻⁴), or 100 nM to 10μM, or 1 to 5 μM or 3 to 70 nM or 10 nM to 1, 2, 3, 4 or 5 μM. Forexample 30 to 70 nM as determined by surface plasmon resonance. In oneembodiment, the first DAB™ (or a DAB™ monomer) binds SA (e.g., HSA) witha K_(D) as determined by surface plasmon resonance of approximately 1,50, 70, 100, 150, 200, 300 nM or 1, 2 or 3 μM. In one embodiment, for adual specific ligand comprising a first anti-SA DAB™ and a second DAB™to TNFR1, the affinity (e.g. K_(D) and/or K_(off) as measured by surfaceplasmon resonance, e.g. using BIACORE™) of the second DAB™ for itstarget is from 1 to 100000 times (e.g., 100 to 100000, or 1000 to100000, or 10000 to 100000 times) the affinity of the first DAB™ for SA.In one embodiment, the serum albumin is human serum albumin (HSA). Forexample, the first DAB™ binds SA with an affinity of approximately 10μM, while the second DAB™ binds its target with an affinity of 100 pM.In one embodiment, the serum albumin is human serum albumin (HSA). Inone embodiment, the first DAB™ binds SA (e.g., HSA) with a K_(D) ofapproximately 50, for example 70, 100, 150 or 200 nM. Details of dualspecific ligands are found in WO03002609, WO04003019 and WO04058821.

The ligands of the invention can in one embodiment comprise a DAB™ thatbinds serum albumin (SA) with a K_(D) as determined by surface plasmonresonance of 1 nM to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 100,200, 300, 400 or 500 μM (i.e., ×10⁻⁹ to 5×10⁻⁴), or 100 nM to 10 μM, or1 to 5 μM or 3 to 70 nM or 10 nM to 1, 2, 3, 4 or 5 μM. For example 30to 70 nM as determined by surface plasmon resonance. In one embodiment,the first DAB™ (or a DAB™ monomer) binds SA (e.g., HSA) with a K_(D) asdetermined by surface plasmon resonance of approximately 1, 50, 70, 100,150, 200, 300 nM or 1, 2 or 3 μM. In one embodiment, the first andsecond DAB™s are linked by a linker, for example a linker of from 1 to 4amino acids or from 1 to 3 amino acids, or greater than 3 amino acids orgreater than 4, 5, 6, 7, 8, 9, 10, 15 or 20 amino acids. In oneembodiment, a longer linker (greater than 3 amino acids) is used toenhance potency (K_(D) of one or both DAB™s in the antagonist).

In particular embodiments of the ligands and antagonists, the DAB™ bindshuman serum albumin and competes for binding to albumin with a DAB™selected from the group consisting of

MSA-16, MSA-26 (See WO04003019 for disclosure of these sequences, whichsequences and their nucleic acid counterpart are incorporated herein byreference and form part of the disclosure of the present text),

DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ IDNO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4(SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480),DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO:483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-22 (SEQ ID NO: 489),DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ IDNO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27(SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497),DOM7r-14 (SEQ ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ IDNO: 500), DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19(SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ IDNO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510), DOM7r-27(SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ ID NO: 513),DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515), DOM7r-32 (SEQ IDNO: 516), DOM7r-33 (SEQ ID NO: 517) (See WO2007080392 for disclosure ofthese sequences, which sequences and their nucleic acid counterpart areincorporated herein by reference and form part of the disclosure of thepresent text; the SEQ ID NO:s in this paragraph are those that appear inWO2007080392),

dAb8 (dAb10), dAb10, dAb36, dAb7r20 (DOM7r20), dAb7r21 (DOM7r21),dAb7r22 (DOM7r22), dAb7r23 (DOM7r23), dAb7r24 (DOM7r24), dAb7r25(DOM7r25), dAb7r26 (DOM7r26), dAb7r27 (DOM7r27), dAb7r28 (DOM7r28),dAb7r29 (DOM7r29), dAb7r29 (DOM7r29), dAb7r31 (DOM7r31), dAb7r32(DOM7r32), dAb7r33 (DOM7r33), dAb7r33 (DOM7r33), dAb7h22 (DOM7h22),dAb7h23 (DOM7h23), dAb7h24 (DOM7h24), dAb7h25 (DOM7h25), dAb7h26(DOM7h26), dAb7h27 (DOM7h27), dAb7h30 (DOM7h30), dAb7h31 (DOM7h31), dAb2(dAbs 4,7,41), dAb4, dAb7, dAb11, dAb12 (dAb7 m12), dAb13 (dAb 15),dAb15, dAb16 (dAb21, dAb7m16), dAb17, dAb18, dAb19, dAb21, dAb22, dAb23,dAb24, dAb25 (dAb26, dAb7m26), dAb27, dAb30 (dAb35), dAb31, dAb33,dAb34, dAb35, dAb38 (dAb54), dAb41, dAb46 (dAbs 47, 52 and 56), dAb47,dAb52, dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1(DOM 7r1), dAb7r3 (DOM7r3), dAb7r4 (DOM7r4), dAb7r5 (DOM7r5), dAb7r7(DOM7r7), dAb7r8 (DOM7r8), dAb7r13 (DOM7r13), dAb7r14 (DOM7r14), dAb7r15(DOM7r15), dAb7r16 (DOM7r16), dAb7r17 (DOM7r17), dAb7r18 (DOM7r18),dAb7r19 (DOM7r19), dAb7h1 (DOM7h1), dAb7h2 (DOM7h2), dAb7h6 (DOM7h6),dAb7h7 (DOM7h7), dAb7h8 (DOM7h8), dAb7h9 (DOM7h9), dAb7h10 (DOM7h10),dAb7h11 (DOM7h11), dAb7h12 (DOM7h12), dAb7h13 (DOM7h13), dAb7h14(DOM7h14), dAb7p1 (DOM7p1), and dAb7p2 (DOM7p2) (see PCT/GB2008/000453filed 8 Feb. 2008 for disclosure of these sequences, which sequences andtheir nucleic acid counterpart are incorporated herein by reference andform part of the disclosure of the present text). Alternative names areshown in brackets after the DAB™, e.g. dAb8 has an alternative namewhich is dAb10 i.e. dAb8 (dAb10). These sequences are also set out inFIGS. 51a and b.

In certain embodiments, the DAB™ binds human serum albumin and comprisesan amino acid sequence that has at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%amino acid sequence identity with the amino acid sequence of a DAB™selected from the group consisting of

MSA-16, MSA-26,

DOM7m-16 (SEQ ID NO: 473), DOM7m-12 (SEQ ID NO: 474), DOM7m-26 (SEQ IDNO: 475), DOM7r-1 (SEQ ID NO: 476), DOM7r-3 (SEQ ID NO: 477), DOM7r-4(SEQ ID NO: 478), DOM7r-5 (SEQ ID NO: 479), DOM7r-7 (SEQ ID NO: 480),DOM7r-8 (SEQ ID NO: 481), DOM7h-2 (SEQ ID NO: 482), DOM7h-3 (SEQ ID NO:483), DOM7h-4 (SEQ ID NO: 484), DOM7h-6 (SEQ ID NO: 485), DOM7h-1 (SEQID NO: 486), DOM7h-7 (SEQ ID NO: 487), DOM7h-22 (SEQ ID NO: 489),DOM7h-23 (SEQ ID NO: 490), DOM7h-24 (SEQ ID NO: 491), DOM7h-25 (SEQ IDNO: 492), DOM7h-26 (SEQ ID NO: 493), DOM7h-21 (SEQ ID NO: 494), DOM7h-27(SEQ ID NO: 495), DOM7h-8 (SEQ ID NO: 496), DOM7r-13 (SEQ ID NO: 497),DOM7r-14 (SEQ ID NO: 498), DOM7r-15 (SEQ ID NO: 499), DOM7r-16 (SEQ IDNO: 500), DOM7r-17 (SEQ ID NO: 501), DOM7r-18 (SEQ ID NO: 502), DOM7r-19(SEQ ID NO: 503), DOM7r-20 (SEQ ID NO: 504), DOM7r-21 (SEQ ID NO: 505),DOM7r-22 (SEQ ID NO: 506), DOM7r-23 (SEQ ID NO: 507), DOM7r-24 (SEQ IDNO: 508), DOM7r-25 (SEQ ID NO: 509), DOM7r-26 (SEQ ID NO: 510), DOM7r-27(SEQ ID NO: 511), DOM7r-28 (SEQ ID NO: 512), DOM7r-29 (SEQ ID NO: 513),DOM7r-30 (SEQ ID NO: 514), DOM7r-31 (SEQ ID NO: 515), DOM7r-32 (SEQ IDNO: 516), DOM7r-33 (SEQ ID NO: 517) (the SEQ ID NO:s in this paragraphare those that appear in WO2007080392),

dAb8, dAb10, dAb36, dAb7r20, dAb7r21, dAb7r22, dAb7r23, dAb7r24,dAb7r25, dAb7r26, dAb7r27, dAb7r28, dAb7r29, dAb7r30, dAb7r31, dAb7r32,dAb7r33, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27,dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15, dAb16,dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26, dAb27,dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47, dAb52,dAb53, dAb54, dAb55, dAb56, dAb7m12, dAb7m16, dAb7m26, dAb7r1, dAb7r3,dAb7r4, dAb7r5, dAb7r7, dAb7r8, dAb7r13, dAb7r14, dAb7r15, dAb7r16,dAb7r17, dAb7r18, dAb7r19, dAb7h1, dAb7h2, dAb7h6, dAb7h7, dAb7h8,dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13, dAb7h14, dAb7p1, and dAb7p2.

For example, the DAB™ that binds human serum albumin can comprise anamino acid sequence that has at least about 90%, or at least about 95%,or at least about 96%, or at least about 97%, or at least about 98%, orat least about 99% amino acid sequence identity with DOM7h-2 (SEQ IDNO:482), DOM7h-3 (SEQ ID NO:483), DOM7h-4 (SEQ ID NO:484), DOM7h-6 (SEQID NO:485), DOM7h-1 (SEQ ID NO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8(SEQ ID NO:496), DOM7r-13 (SEQ ID NO:497), DOM7r-14 (SEQ ID NO:498),DOM7h-22 (SEQ ID NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ IDNO:491), DOM7h-25 (SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493), DOM7h-21(SEQ ID NO:494), DOM7h-27 (SEQ ID NO:495) (the SEQ ID NO:s in thisparagraph are those that appear in WO2007080392),

dAb8, dAb10, dAb36, dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26,dAb7h27, dAb7h30, dAb7h31, dAb2, dAb4, dAb7, dAb11, dAb12, dAb13, dAb15,dAb16, dAb17, dAb18, dAb19, dAb21, dAb22, dAb23, dAb24, dAb25, dAb26,dAb27, dAb30, dAb31, dAb33, dAb34, dAb35, dAb38, dAb41, dAb46, dAb47,dAb52, dAb53, dAb54, dAb55, dAb56, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13 and dAb7h14.

In certain embodiments, the DAB™ binds human serum albumin and comprisesan amino acid sequence that has at least about 80%, or at least about85%, or at least about 90%, or at least about 95%, or at least about96%, or at least about 97%, or at least about 98%, or at least about 99%amino acid sequence identity with the amino acid sequence of a DAB™selected from the group consisting of

DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ IDNO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496), DOM7h-22 (SEQID NO:489), DOM7h-23 (SEQ ID NO:490), DOM7h-24 (SEQ ID NO:491), DOM7h-25(SEQ ID NO:492), DOM7h-26 (SEQ ID NO:493), DOM7h-21 (SEQ ID NO:494),DOM7h-27 (SEQ ID NO:495) (the SEQ ID NO:s in this paragraph are thosethat appear in WO2007080392),

dAb7h21, dAb7h22, dAb7h23, Ab7h24, Ab7h25, Ab7h26, dAb7h27, dAb7h30,dAb7h31, dAb2, dAb4, dAb7, dAb38, dAb41, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13 and dAb7h14.

In more particular embodiments, the DAB™ is a V_(κ) DAB™ that bindshuman serum albumin and has an amino acid sequence selected from thegroup consisting of

DOM7h-2 (SEQ ID NO:482), DOM7h-6 (SEQ ID NO:485), DOM7h-1 (SEQ IDNO:486), DOM7h-7 (SEQ ID NO:487), DOM7h-8 (SEQ ID NO:496) (the SEQ IDNO:s in this paragraph are those that appear in WO2007080392),

dAb2, dAb4, dAb7, dAb38, dAb41, dAb54, dAb7h1, dAb7h2, dAb7h6, dAb7h7,dAb7h8, dAb7h9, dAb7h10, dAb7h11, dAb7h12, dAb7h13 and dAb7h14.

In more particular embodiments, the DAB™ is a V_(H) DAB™ that bindshuman serum albumin and has an amino acid sequence selected from dAb7h30and dAb7h31.

In more particular embodiments, the DAB™ is dAb7h11 or dAb7h14.

In other embodiments, the DAB™, ligand or antagonist binds human serumalbumin and comprises one, two or three of the CDRs of any of theforegoing amino acid sequences, e.g. one, two or three of the CDRs ofdAb7h11 or dAb7h14.

Suitable Camelid V_(HH) that bind serum albumin include those disclosedin WO 2004/041862 (Ablynx N.V.) and in WO2007080392 (which V_(HH)sequences and their nucleic acid counterpart are incorporated herein byreference and form part of the disclosure of the present text), such asSequence A (SEQ ID NO:518), Sequence B (SEQ ID NO:519), Sequence C (SEQID NO:520), Sequence D (SEQ ID NO:521), Sequence E (SEQ ID NO:522),Sequence F (SEQ ID NO:523), Sequence G (SEQ ID NO:524), Sequence H (SEQID NO:525), Sequence I (SEQ ID NO:526), Sequence J (SEQ ID NO:527),Sequence K (SEQ ID NO:528), Sequence L (SEQ ID NO:529), Sequence M (SEQID NO:530), Sequence N (SEQ ID NO:531), Sequence 0 (SEQ ID NO:532),Sequence P (SEQ ID NO:533), Sequence Q (SEQ ID NO:534), these sequencenumbers correspond to those cited in WO2007080392 or WO 2004/041862(Ablynx N.V.). In certain embodiments, the Camelid V_(HH) binds humanserum albumin and comprises an amino acid sequence that has at leastabout 80%, or at least about 85%, or at least about 90%, or at leastabout 95%, or at least about 96%, or at least about 97%, or at leastabout 98%, or at least about 99% amino acid sequence identity with ALB1disclosed in WO2007080392 or any one of SEQ ID NOS:518-534, thesesequence numbers corresponding to those cited in WO2007080392 or WO2004/041862.

In some embodiments, the ligand or antagonist comprises an anti-serumalbumin DAB™ that competes with any anti-serum albumin DAB™ disclosedherein for binding to serum albumin (e.g., human serum albumin).

In an alternative embodiment, the antagonist or ligand comprises abinding moiety specific for TNFR1 (e.g., human TNFR1), wherein themoiety comprises non-immunoglobulin sequences as described in co-pendingapplication PCT/GB2008/000453 filed 8 Feb. 2008, the disclosure of thesebinding moieties, their methods of production and selection (e.g., fromdiverse libraries) and their sequences are incorporated herein byreference as part of the disclosure of the present text)

Conjugation to a Half-Life Extending Moiety (e.g., Albumin)

In one embodiment, a (one or more) half-life extending moiety (e.g.,albumin, transferrin and fragments and analogues thereof) is conjugatedor associated with the TNFR1-binding polypeptide, DAB™ or antagonist ofthe invention. Examples of suitable albumin, albumin fragments oralbumin variants for use in a TNFR1-binding format are described in WO2005077042, which disclosure is incorporated herein by reference andforms part of the disclosure of the present text. In particular, thefollowing albumin, albumin fragments or albumin variants can be used inthe present invention:

-   -   SEQ ID NO:1 (as disclosed in WO 2005077042, this sequence being        explicitly incorporated into the present disclosure by        reference);    -   Albumin fragment or variant comprising or consisting of amino        acids 1-387 of SEQ ID NO:1 in WO 2005077042;    -   Albumin, or fragment or variant thereof, comprising an amino        acid sequence selected from the group consisting of: (a) amino        acids 54 to 61 of SEQ ID NO:1 in WO 2005077042; (b) amino acids        76 to 89 of SEQ ID NO:1 in WO 2005077042; (c) amino acids 92 to        100 of SEQ ID NO:1 in WO 2005077042; (d) amino acids 170 to 176        of SEQ ID NO:1 in WO 2005077042; (e) amino acids 247 to 252 of        SEQ ID NO:1 in WO 2005077042; (f) amino acids 266 to 277 of SEQ        ID NO:1 in WO 2005077042; (g) amino acids 280 to 288 of SEQ ID        NO:1 in WO 2005077042; (h) amino acids 362 to 368 of SEQ ID NO:1        in WO 2005077042; (i) amino acids 439 to 447 of SEQ ID NO:1 in        WO 2005077042 (j) amino acids 462 to 475 of SEQ ID NO:1 in WO        2005077042; (k) amino acids 478 to 486 of SEQ ID NO:1 in WO        2005077042; and (l) amino acids 560 to 566 of SEQ ID NO:1 in WO        2005077042.

Further examples of suitable albumin, fragments and analogs for use in aTNFR1-binding format are described in WO 03076567, which disclosure isincorporated herein by reference and which forms part of the disclosureof the present text. In particular, the following albumin, fragments orvariants can be used in the present invention:

-   -   Human serum albumin as described in WO 03076567, e.g., in FIG. 3        (this sequence information being explicitly incorporated into        the present disclosure by reference);    -   Human serum albumin (HA) consisting of a single non-glycosylated        polypeptide chain of 585 amino acids with a formula molecular        weight of 66,500 (See, Meloun, et al., FEBS Letters 58:136        (1975); Behrens, et al., Fed. Proc. 34:591 (1975); Lawn, et al.,        Nucleic Acids Research 9:6102-6114 (1981); Minghetti, et al., J.        Biol. Chem. 261:6747 (1986));    -   A polymorphic variant or analog or fragment of albumin as        described in Weitkamp, et al., Ann. Hum. Genet. 37:219 (1973);    -   An albumin fragment or variant as described in EP 322094, e.g.,        HA(1-373, HA(1-388), HA(1-389), HA(1-369), and HA(1-419) and        fragments between 1-369 and 1-419;    -   An albumin fragment or variant as described in EP 399666, e.g.,        HA(1-177) and HA(1-200) and fragments between HA(1-X), where X        is any number from 178 to 199.

Where a (one or more) half-life extending moiety (e.g., albumin,transferrin and fragments and analogues thereof) is used to format theTNFR1-binding polypeptides, DAB™s and antagonists of the invention, itcan be conjugated using any suitable method, such as, by direct fusionto the TNFR1-binding moiety (e.g., anti-TNFR1 DAB™), for example byusing a single nucleotide construct that encodes a fusion protein,wherein the fusion protein is encoded as a single polypeptide chain withthe half-life extending moiety located N- or C-terminally to the TNFR1binding moiety. Alternatively, conjugation can be achieved by using apeptide linker between moeities, e.g., a peptide linker as described inWO 03076567 or WO 2004003019 (these linker disclosures beingincorporated by reference in the present disclosure to provide examplesfor use in the present invention). Typically, a polypeptide thatenhances serum half-life in vivo is a polypeptide which occurs naturallyin vivo and which resists degradation or removal by endogenousmechanisms which remove unwanted material from the organism (e.g.,human). For example, a polypeptide that enhances serum half-life in vivocan be selected from proteins from the extracellular matrix, proteinsfound in blood, proteins found at the blood brain barrier or in neuraltissue, proteins localized to the kidney, liver, lung, heart, skin orbone, stress proteins, disease-specific proteins, or proteins involvedin Fc transport.

In embodiments of the invention described throughout this disclosure,instead of the use of an anti-TNFR1 “DAB™” in an antagonist or ligand ofthe invention, it is contemplated that the skilled addressee can use apolypeptide or domain that comprises one or more or all 3 of the CDRs ofa DAB™ of the invention that binds TNFR1 (e.g., CDRs grafted onto asuitable protein scaffold or skeleton, e.g. an affibody, an SpAscaffold, an LDL receptor class A domain or an EGF domain) Thedisclosure as a whole is to be construed accordingly to providedisclosure of antagonists using such domains in place of a DAB™. In thisrespect, see PCT/GB2008/000453 filed 8 Feb. 2008, the disclosure ofwhich is incorporated by reference).

In one embodiment, therefore, an antagonist of the invention comprisesan immunoglobulin single variable domain or domain antibody (DAB™) thathas binding specificity for TNFR1 or the complementarity determiningregions of such a DAB™ in a suitable format. The antagonist can be apolypeptide that consists of such a DAB™, or consists essentially ofsuch a DAB™. The antagonist can be a polypeptide that comprises a DAB™(or the CDRs of a DAB™) in a suitable format, such as an antibody format(e.g., IgG-like format, scFv, Fab, Fab′, F(ab′)₂), or a dual specificligand that comprises a DAB™ that binds TNFR1 and a second DAB™ thatbinds another target protein, antigen or epitope (e.g., serum albumin).

Polypeptides, DAB™s and antagonists according to the invention can beformatted as a variety of suitable antibody formats that are known inthe art, such as, IgG-like formats, chimeric antibodies, humanizedantibodies, human antibodies, single chain antibodies, bispecificantibodies, antibody heavy chains, antibody light chains, homodimers andheterodimers of antibody heavy chains and/or light chains,antigen-binding fragments of any of the foregoing (e.g., a Fv fragment(e.g., single chain Fv (scFv), a disulfide bonded Fv), a Fab fragment, aFab′ fragment, a F(ab′)₂ fragment), a single variable domain (e.g.,V_(H), V_(L)), a DAB™, and modified versions of any of the foregoing(e.g., modified by the covalent attachment of polyalkylene glycol (e.g.,polyethylene glycol, polypropylene glycol, polybutylene glycol) or othersuitable polymer).

In some embodiments, the invention provides a ligand (e.g., ananti-TNFR1 antagonist) that is an IgG-like format. Such formats have theconventional four chain structure of an IgG molecule (2 heavy chains andtwo light chains), in which one or more of the variable regions (V_(H)and or V_(L)) have been replaced with a DAB™ of the invention. In oneembodiment, each of the variable regions (2 V_(H) regions and 2 V_(L)regions) is replaced with a DAB™ or single variable domain, at least oneof which is an anti-TNFR1 DAB™ according to the invention. The DAB™(s)or single variable domain(s) that are included in an IgG-like format canhave the same specificity or different specificities. In someembodiments, the IgG-like format is tetravalent and can have one(anti-TNFR1 only), two (e.g., anti-TNFR1 and anti-SA), three or fourspecificities. For example, the IgG-like format can be monospecific andcomprises 4 DAB™s that have the same specificity; bispecific andcomprises 3 DAB™s that have the same specificity and another DAB™ thathas a different specificity; bispecific and comprise two DAB™s that havethe same specificity and two DAB™s that have a common but differentspecificity; trispecific and comprises first and second DAB™s that havethe same specificity, a third DAB™ with a different specificity and afourth DAB™ with a different specificity from the first, second andthird DAB™s; or tetraspecific and comprise four DAB™s that each have adifferent specificity. Antigen-binding fragments of IgG-like formats(e.g., Fab, F(ab′)₂, Fab′, Fv, scF_(V)) can be prepared. In oneembodiment, the IgG-like formats or antigen-binding fragments thereof donot crosslink TNFR1, for example, the format may be monovalent forTNFR1. If complement activation and/or antibody dependent cellularcytotoxicity (ADCC) function is desired, the ligand can be an IgG1-likeformat. If desired, the IgG-like format can comprise a mutated constantregion (variant IgG heavy chain constant region) to minimize binding toFc receptors and/or ability to fix complement. (see e.g. Winter et al.,GB 2,209,757 B; Morrison et al., WO 89/07142; Morgan et al., WO94/29351, Dec. 22, 1994).

The ligands of the invention (polypeptides, DAB™s and antagonists) canbe formatted as a fusion protein that contains a first immunoglobulinsingle variable domain that is fused directly to a second immunoglobulinsingle variable domain. If desired such a format can further comprise ahalf-life extending moiety. For example, the ligand can comprise a firstimmunoglobulin single variable domain that is fused directly to a secondimmunoglobulin single variable domain that is fused directly to animmunoglobulin single variable domain that binds serum albumin.

Generally the orientation of the polypeptide domains that have a bindingsite with binding specificity for a target, and whether the ligandcomprises a linker, is a matter of design choice. However, someorientations, with or without linkers, may provide better bindingcharacteristics than other orientations. All orientations (e.g.,dAb1-linker-dAb2; dAb2-linker-dAb1) are encompassed by the invention areligands that contain an orientation that provides desired bindingcharacteristics can be easily identified by screening.

Polypeptides and DAB™s according to the invention, including DAB™monomers, dimers and trimers, can be linked to an antibody Fc region,comprising one or both of C_(H)2 and C_(H)3 domains, and optionally ahinge region. For example, vectors encoding ligands linked as a singlenucleotide sequence to an Fc region may be used to prepare suchpolypeptides.

The invention moreover provides dimers, trimers and polymers of theaforementioned DAB™ monomers.

Codon Optimised Sequences

As described above, embodiments of the invention provide codon optimizednucleotide sequences encoding polypeptides and variable domains of theinvention. As shown in the following illustration, codon optimizedsequences of about 70% identity can be produced that encode for the samevariable domain (in this case the variable domain amino acid sequence isidentical to DOM1h-131-206). In this instance, the sequences wereoptimized for expression by Pichia pastoris (codon optimized sequences1-3) or E. coli (codon optimized sequences 4 and 5).

We performed a calculation taking into account the degeneracy in thegenetic code and maximised the number of nucleotide changes within eachdegenerate codon encoded by the nucleotide sequence of DOM1h-131-206 asshown in FIG. 19 and a theoretical nucleotide sequence which stillencodes a variable domain that is identical to DOM1h-131-206. Thecalculation revealed that the theoretical sequence would have only 57%identity to the nucleotide sequence of DOM1h-131-206 as shown in FIG.19.

DNA SequenceGaggttcaattgttggaatccggtggtggattggttcaacctggtggttctttgagattgtcctgtgctg(SEQ ID NO: 223)cttccggttttactttcgctcacgagactatggtttgggttagacaggctccaggtaaaggattggaatgggtttcccacattccaccagatggtcaagatccattctacgctgactccgttaagggaagattcactatctccagagacaactccaagaacactttgtacttgcagatgaactccttgagagctgaggatactgctgtttaccactgtgctttgttgccaaagagaggaccttggtttgattactggggacagggaactttggttactgtttcttcc Corresponding AA SequenceEvq1lesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrfti(SEQ ID NO: 224) srdnskntlylqmnslraedtavyhcallpkrgpwfdywgqgtivtvss •74.1% nucleotide sequence identity to WT sequence (nucleic acid sequencesshown below correspond to SEQ ID NO:s 225-228)1                                               50Dom1h-131-206 Codon Optimised (1)GAGGTTCAATTGTTGGAATCCGGTGGTGGATTGGTTCAACCTGGTGGTTC Dom1h-131-206 WT (1)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC Consensus (1)GAGGT CA  TGTTGGA TC GG GG GG TTGGT CA CCTGG GG TC51                                             100Dom1h-131-206 Codon Optimised (51)TTTGAGATTGTCCTGTGCTGCTTCCGGTTTTACTTTCGCTCACGAGACTA Dom1h-131-206 WT (51)CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCGCATGAGACGA Consensus (51)  TG G  T TCCTGTGC GC TCCGG TT AC TT GC CA GAGAC A101                                            150Dom1h-131-206 Codon Optimised (101)TGGTTTGGGTTAGACAGGCTCCAGGTAAAGGATTGGAATGGGTTTCCCAC Dom1h-131-206 WT(101) TGGTGTGGGTCCGCCAGGCACCAGGGAAGGGTCTAGAGTGGGTCTCACAT Consensus (101)TGGT TGGGT  G CAGGC CCAGG AA GG  T GA TGGGT TC CA151                                            200Dom1h-131-206 Codon Optimised (151)ATTCCACCAGATGGTCAAGATCCATTCTACGCTGACTCCGTTAAGGGAAG Dom1h-131-206 WT(151) ATTCCCCCGGATGGTCAGGATCCCTTCTACGCAGACTCCGTGAAGGGCCG Consensus (151)ATTCC CC GATGGTCA GATCC TTCTACGC GACTCCGT AAGGG  G201                                            250Dom1h-131-206 Codon Optimised (201)ATTCACTATCTCCAGAGACAACTCCAAGAACACTTTGTACTTGCAGATGA Dom1h-131-206 WT(201) GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTATATCTGCAAATGA Consensus (201)TTCAC ATCTCC G GACAA TCCAAGAACAC  T TA  TGCA ATGA251                                            300Dom1h-131-206 Codon Optimised (251)ACTCCTTGAGAGCTGAGGATACTGCTGTTTACCACTGTGCTTTGTTGCCA Dom1h-131-206 WT(251) ACAGCCTGCGTGCCGAGGACACAGCGGTATATCACTGTGCGCTGCTTCCT Consensus (251)AC  C TG G GC GAGGA AC GC GT TA CACTGTGC  TG T CC301                                            350Dom1h-131-206 Codon Optimised (301)AAGAGAGGACCTTGGTTTGATTACTGGGGACAGGGAACTTTGGTTACTGT Dom1h-131-206 WT(301) AAGAGGGGGCCTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGT Consensus (301)AAGAG GG CCTTGGTTTGA TACTGGGG CAGGGAAC  TGGT AC GT 351       363Dom1h-131-206 Codon Optimised (351) TTCTTCCTAATGA Dom1h-131-206 WT (351)CTCGAGC------ Consensus (351)  TC  C Codon Optimised Sequence 2DNA SequenceGagaaaagagaggttcaattgcttgaatctggaggaggtttggtccagccaggagggtcccttcgactaa(SEQ ID NO: 229)gttgtgctgccagtgggtttacgtttgctcatgaaactatggtatgggtccgacaggcacctggtaaaggtcttgaatgggtttcacatatccctccagacggtcaagacccattttacgctgattccgtgaaaggcagatttacaatttcacgagataattctaaaaacaccttgtacttacaaatgaactcattgagagctgaggacactgcagtttatcactgcgctttactaccaaaacgtggaccttggtttgattattggggccaaggtacgttagtgactgttagttct Corresponding AA SequenceEkrevq1lesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgr(SEQ ID NO: 230) ftisrdnskntlylqmnslraedtavyhcallpkrgpwfdywgqgtivtvss •71.1% nucleotide sequence identoty to WT sequence (nucleic acid sequencesshown below correspond to SEQ ID NO:s 231-233)1                                               50 Dom1h-131-206 WT (1)---------GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC Pichia MFa 206 DAB ™only (1) GAGAAAAGAGAGGTTCAATTGCTTGAATCTGGAGGAGGTTTGGTCCAGCC Consensus(1)          GAGGT CA  TG T GA TCTGG GGAGG TTGGT CAGCC51                                             100 Dom1h-131-206 WT (42)TGGGGGGTCCCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCGC Pichia MFa 206 DAB ™only (51) AGGAGGGTCCCTTCGACTAAGTTGTGCTGCCAGTGGGTTTACGTTTGCTC Consensus(51)  GG GGGTCCCT CG CT    TGTGC GCC   GG TT AC TTTGC C101                                            150 Dom1h-131-206 WT (92)ATGAGACGATGGTGTGGGTCCGCCAGGCACCAGGGAAGGGTCTAGAGTGG Pichia MFa 206 DAB ™only (101) ATGAAACTATGGTATGGGTCCGACAGGCACCTGGTAAAGGTCTTGAATGG Consensus(101) ATGA AC ATGGT TGGGTCCG CAGGCACC GG AA GGTCT GA TGG151                                            200 Dom1h-131-206 WT(142) GTCTCACATATTCCCCCGGATGGTCAGGATCCCTTCTACGCAGACTCCGTPichia MFa 206 DAB ™ only (151)GTTTCACATATCCCTCCAGACGGTCAAGACCCATTTTACGCTGATTCCGT Consensus (151)GT TCACATAT CC CC GA GGTCA GA CC TT TACGC GA TCCGT201                                            250 Dom1h-131-206 WT(192) GAAGGGCCGGTTCACCATCTCCCGCGACAATTCCAAGAACACGCTATATCPichia MFa 206 DAB ™ only (201)GAAAGGCAGATTTACAATTTCACGAGATAATTCTAAAAACACCTTGTACT Consensus (201)GAA GGC G TT AC AT TC CG GA AATTC AA AACAC  T TA251                                            300 Dom1h-131-206 WT(242) TGCAAATGAACAGCCTGCGTGCCGAGGACACAGCGGTATATCACTGTGCGPichia MFa 206 DAB ™ only (251)TACAAATGAACTCATTGAGAGCTGAGGACACTGCAGTTTATCACTGCGCT Consensus (251)T CAAATGAAC    TG G GC GAGGACAC GC GT TATCACTG GC301                                            350 Dom1h-131-206 WT(292) CTGCTTCCTAAGAGGGGGCCTTGGTTTGACTACTGGGGTCAGGGAACCCTPichia MFa 206 DAB ™ only (301)TTACTACCAAAACGTGGACCTTGGTTTGATTATTGGGGCCAAGGTACGTT Consensus (301) T CT CC AA  G GG CCTTGGTTTGA TA TGGGG CA GG AC  T 351           367Dom1h-131-206 WT (342) GGICACCGTCTCGAGC- Phchha MFa 206 DAB ™ only (351)AGTGACTGT-TAGTTCT Consensus (351)  GT AC GT T G  CCodon Optimised Sequence 3 DNA SequenceGaagtgcagcttcttgaaagtggtggagggctagtgcagccagggggatctttaagattatcatgcgctg(SEQ ID NO: 234)ccagtggatttacttttgctcacgagacgatggtctgggtgagacaagctcctggaaaaggtttagagtgggtttctcacattccacctgatggtcaagatcctttctacgcagattccgtcaaaggaagatttactatctccagagataatagtaaaaacactttgtacctacagatgaactcacttagagccgaagataccgctgtgtaccactgcgccttgttgccaaagagaggtccttggttcgattactggggtcagggtactctggttacagtctcatct Corresponding AA SequenceEvq1lesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrfti(SEQ ID NO: 235) srdnskntlylqmnslraedtavyhcallpkrgpwfdywgqgtivtvss •72.6% nucleotide sequence identity to WT sequence (nucleic acid sequencesshown below correspond to SEQ ID NO:s 236-240)1                                               50 Dom1h-131-206 WT (1)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC Pichia Pre 206 DAB ™only (1) GAAGTGCAGCTTCTTGAAAGTGGTGGAGGGCTAGTGCAGCCAGGGGGATC Consensus(1) GA GTGCAGCT  T GA   TGG GGAGG  T GT CAGCC GGGGG TC51                                             100 Dom1h-131-206 WT (51)CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCGCATGAGACGA Pichia Pre 206 DAB ™only (51) TTTAAGATTATCATGCGCTGCCAGTGGATTTACTTTTGCTCACGAGACGA Consensus(51)  T  G  T TC TG GC GCC   GGATT AC TTTGC CA GAGACGA101                                            150 Dom1h-131-206 WT(101) TGGTGTGGGTCCGCCAGGCACCAGGGAAGGGTCTAGAGTGGGTCTCACATPichia Pre 206 DAB ™ only (101)TGGTCTGGGTGAGACAAGCTCCTGGAAAAGGTTTAGAGTGGGTTTCTCAC Consensus (101)TGGT TGGGT  G CA GC CC GG AA GGT TAGAGTGGGT TC CA151                                            200 Dom1h-131-206 WT(151) ATTCCCCCGGATGGTCAGGATCCCTTCTACGCAGACTCCGTGAAGGGCCGPichia Pre 206 DAB ™ only (151)ATTCCACCTGATGGTCAAGATCCTTTCTACGCAGATTCCGTCAAAGGAAG Consensus (151)ATTCC CC GATGGTCA GATCC TTCTACGCAGA TCCGT AA GG  G201                                            250 Dom1h-131-206 WT(201) GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTATATCTGCAAATGAPichia Pre 206 DAB ™ only (201)ATTTACTATCTCCAGAGATAATAGTAAAAACACTTTGTACCTACAGATGA Consensus (201)TT AC ATCTCC G GA AAT   AA AACAC  T TA CT CA ATGA251                                            300 Dom1h-131-206 WT(251) ACAGCCTGCGTGCCGAGGACACAGCGGTATATCACTGTGCGCTGCTTCCTPichia Pre 206 DAB ™ only (251)ACTCACTTAGAGCCGAAGATACCGCTGTGTACCACTGCGCCTTGTTGCCA Consensus (251)AC   CT  G GCCGA GA AC GC GT TA CACTG GC  TG T CC301                                            350 Dom1h-131-206 WT(301) AAGAGGGGGCCTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTPichia Pre 206 DAB ™ only (301)AAGAGAGGTCCTTGGTTCGATTACTGGGGTCAGGGTACTCTGGTTACAGT Consensus (301)AAGAG GG CCTTGGTT GA TACTGGGGTCAGGG AC CTGGT AC GT 351 Dom1h-131-206 WT(351) CTCGAGC- Pichla Pre 206 DAB ™ only (351) CTC-ATCT Consensus (351)CTC A C Codon Optimised Sequence 4 DNA SequenceGaagtacaactgctggagagcggtggcggcctggttcaaccgggtggttccctgcgcctgtcctgtgcgg(SEQ ID No; 241)catctggtttcaccttcgcacacgaaaccatggtgtgggttcgccaagctccgggcaaaggcctggaatgggtaagccacattcctccagatggccaggacccattctatgcggattccgttaagggtcgctttaccatttctcgtgataactccaaaaacaccctgtacctgcagatgaactccctgcgcgccgaggatactgcggtgtaccattgtgcgctgctgcctaaacgtggcccgtggttcgattactggggtcagggtactctggtcaccgtaagcagc Corresponding AA SequenceEvgllesggglvgpggslrlscaasgftfahetmvwvrgapgkglewvshippdggdpfyadsvkgrfti(SEQ ID NO: 242) srdnskntlylgmnslraedtavyhcallpkrgpwfdywgggtivtvss •76.5% nucleotide sequence identity to WT sequence (nucleic acid sequencesshown below correspond to SEQ ID NO:s 243-248)1                                               50 Dom1h-131-206 WT (1)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC Ecoli Sec 206 DAB ™only (1) GAAGTACAACTGCTGGAGAGCGGTGGCGGCCTGGTTCAACCGGGTGGTTC Consensus(1) GA GT CA CTG TGGAG   GG GG GGC TGGT CA CC GG GG TC51                                             100 Dom1h-131-206 WT (51)CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCGCATGAGACGA Ecoli Sec 206 DAB ™only (51) CCTGCGCCTGTCCTGTGCGGCATCTGGTTTCACCTTCGCACACGAAACCA Consensus(51) CCTGCG CT TCCTGTGC GC TC GG TTCACCTT GC CA GA AC A101                                            150 Dom1h-131-206 WT(101) TGGTGTGGGTCCGCCAGGCACCAGGGAAGGGTCTAGAGTGGGTCTCACATEcoll Sec 206 DAB ™ only (101)TGGTGTGGGTTCGCCAAGCTCCGGGCAAAGGCCTGGAATGGGTAAGCCAC Consensus (101)TGGTGTGGGT CGCCA GC CC GG AA GG CT GA TGGGT    CA151                                            200 Dom1h-131-206 WT(151) ATTCCCCCGGATGGTCAGGATCCCTTCTACGCAGACTCCGTGAAGGGCCGEcoll Sec 206 DAB ™ only (151)ATTCCTCCAGATGGCCAGGACCCATTCTATGCGGATTCCGTTAAGGGTCG Consensus (151)ATTCC CC GATGG CAGGA CC TTCTA GC GA TCCGT AAGGG CG201                                            250 Dom1h-131-206 WT(201) GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTATATCTGCAAATGAEcoll Sec 206 DAB ™ only (201)CTTTACCATTTCTCGTGATAACTCCAAAAACACCCTGTACCTGCAGATGA Consensus (201)TT ACCAT TC CG GA AA TCCAA AACAC CT TA CTGCA ATGA251                                            300 Dom1h-131 DAB ™ only(251) ACTCCCTGCGCGCCGAGGATACTGCGGTGTACCATTGTGCGCTGCTGCCT Consensus (251)AC  CCTGCG GCCGAGGA AC GCGGT TA CA TGTGCGCTGCT CCT301                                            350 Dom1h-131-206 WT(301) AAGAGGGGGCCTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTEcoll Sec 206 DAB ™ only (301)AAACGTGGCCCGTGGTTCGATTACTGGGGTCAGGGTACTCTGGTCACCGT Consensus (301)AA  G GG CC TGGTT GA TACTGGGGTCAGGG AC CTGGTCACCGT 351 Dom1h-131-206 WT(351) CTCGAGC Ecoll Sec 206 DAB ™ only (351) AAGCAGC Consensus (351)    AGC Codon Optimised Sequence 5 DNA SequenceGaggttcaactgctggaatctggtggtggtctggtacaaccgggtggttccctgcgtctgagctgtgcag(SEQ ID NO: 249)cctctggtttcaccttcgctcatgagaccatggtttgggtacgccaggctccgggtaaaggcctggagtgggtaagccatatccctcctgatggtcaggacccgttctatgctgattccgtcaaaggccgttttaccatttctcgtgacaacagcaaaaacactctgtacctgcaaatgaactccctgcgtgcagaagacacggcggtttatcactgtgcactgctgccaaaacgcggcccttggttcgactactggggccagggtactctggtcactgtatcttct Corresponding AA SequenceEvqllesggglvqpggslrlscaasgftfahetmvwvrqapgkglewvshippdgqdpfyadsvkgrfti(SEQ ID NO: 250) srdnskntlylqmnslraedtavyhcallpkrgpwfdywgqgtivtvss •78.4% nucleotide sequence identity to WT sequence (nucleic acid sequencesshown below correspond to SEQ ID NO:s 251-257)1                                               50 Dom1h-131-206 WT (1)GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTC Ecoll IC 206 DAB ™only (1) GAGGTTCAACTGCTGGAATCTGGTGGTGGTCTGGTACAACCGGGTGGTTC Consensus(1) GAGGT CA CTG TGGA TCTGG GG GG  TGGTACA CC GG GG TC51                                             100 Dom1h-131-206 WT (51)CCTGCGTCTCTCCTGTGCAGCCTCCGGATTCACCTTTGCGCATGAGACGA Ecoll IC 206 DAB ™only (51) CCTGCGTCTGAGCTGTGCAGCCTCTGGTTTCACCTTCGCTCATGAGACCA Consensus(51) CCTGCGTCT   CTGTGCAGCCTC GG TTCACCTT GC CATGAGAC A101                                            150 Dom1h-131-206 WT(101) TGGTGTGGGTCCGCCAGGCACCAGGGAAGGGTCTAGAGTGGGTCTCACATEcoll IC 206 DAB ™ only (101)TGGTTTGGGTACGCCAGGCTCCGGGTAAAGGCCTGGAGTGGGTAAGCCAT Consensus (101)TGGT TGGGT CGCCAGGC CC GG AA GG CT GAGTGGGT    CAT151                                            200 Dom1h-131-206 WT(151) ATTCCCCCGGATGGTCAGGATCCCTTCTACGCAGACTCCGTGAAGGGCCGEcoll IC 206 DAB ™ only (151)ATCCCTCCTGATGGTCAGGACCCGTTCTATGCTGATTCCGTCAAAGGCCG Consensus (151)AT CC CC GATGGTCAGGA CC TTCTA GC GA TCCGT AA GGCCG201                                            250 Dom1h-131-206 WT(201) GTTCACCATCTCCCGCGACAATTCCAAGAACACGCTATATCTGCAAATGAEcoll IC 206 DAB ™ only (201)TTTTACCATTTCTCGTGACAACAGCAAAAACACTCTGTACCTGCAAATGA Consensus (201) TT ACCAT TC CG GACAA   CAA AACAC CT TA CTGCAAATGA251                                            300 Dom1h-131-206 WT(251) ACAGCCTGCGTGCCGAGGACACAGCGGTATATCACTGTGCGCTGCTTCCTEcoll IC 206 DAB ™ only (251)ACTCCCTGCGTGCAGAAGACACGGCGGTTTATCACTGTGCACTGCTGCCA Consensus (251)AC  CCTGCGTGC GA GACAC GCGGT TATCACTGTGC CTGCT CC301                                            350 Dom1h-131-206 WT(301) AAGAGGGGGCCTTGGTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTEcoli IC 206 DAB ™ only (301)AAACGCGGCCCTTGGTTCGACTACTGGGGCCAGGGTACTCTGGTCACTGT Consensus (301)AA  G GG CCTTGGTT GACTACTGGGG CAGGG AC CTGGTCAC GT 351 Dom1h-131-206 WT(351) CTCGAGC Ecoli IC 206 DAB ™ only (351) ATCTTCT Consensus (351)  TC

EXEMPLIFICATION Example A Lead Selection & Characterisation of DomainAntibodies to Human TNFR1

Domain antibodies generated were derived from phage libraries. Bothsoluble selections and panning to passively absorbed human TNFR1 wereperformed according to the relevant standard methods. Human TNFR1 waspurchased as a soluble recombinant protein either from R&D systems (CatNo 636-R1-025/CF) or Peprotech (Cat no. 310-07) and either used directly(in the case of passive selections) or after biotinylation usingcoupling via primary amines followed by quality control of its activityin a biological assay and analysis of its MW and extent of biotinylationby mass spectrometry. Typically 3 rounds of selection were performedutilising decreasing levels of antigen in every next round.

Outputs from selections were screened by phage ELISA for the presence ofanti-TNFR1 binding clones. DNA was isolated from these phage selectionsand subcloned into a expression vector for expression of soluble DAB™fragments. Soluble DAB™ fragments were expressed in 96-well plates andthe supernatants were used to screen for the presence of anti-TNFR1binding DAB™s, either using a direct binding ELISA with anti-c-mycdetection or BIACORE™ using a streptavidin/biotinylated TNFR1BIACORE™_chip and ranked according to off-rates.

The lead molecules, described below, were derived from the parentalDAB™, designated DOM1h-131 (disclosed in WO2006038027). This moleculewas selected from the phage display library after 3 rounds of selectionsusing 60 nM of biotinylated antigen. Streptavidin or NEUTRAVIDIN™ coatedDYNABEADS™ were alternated as capture reagents in each round ofselection to prevent selection of binders against either streptavidin orNEUTRAVIDIN™. The potency of the lead DOM1h-131 at this stage was in thelow micromolar range as determined in the MRC-5 fibroblast/IL-8 releasecell assay. The binding kinetics as determined by BIACORE™ typicallydisplayed fast-on/fast-off rates. E. coli expression levels of thisDOM1h-131 lead molecule, as a C-terminally myc tagged monomer were inthe region of 8 mg/l.

Affinity Maturation of Leads:

DOM1h-131 was taken forward into affinity maturation to generate mutantswith higher potency and improved biophysical characteristics (see FIG. 3for amino acid sequences of DOM1h-131 derived leads). After generationof an error-prone library (average number of 1 amino acid change perDAB™ sequence, library size 8×10⁷) using an error-prone PCR polymerase(GENEMORPH II™, Stratagene), seven rounds of selection utilising theseerror-prone libraries were performed. This strategy led to the isolationof clone DOM1h-131-8, a molecule where 4 amino acid changes (one inframework 1 (FR1), one in CDR1, one in CDR3 and one in FR4) gave anapproximate 100-fold improvement in potency as measured by the MRC-5cell assay (˜4 nM). In this assay MRC-5 cells were incubated with thetest samples for one hour then TNF-α (200 pg/ml) was added. After anovernight incubation IL-8 release was determined using an IL-8 ABI 8200™cellular detection assay (FMAT™). A TNF-α dose curve was included ineach experiment. The concentration of TNF-α used to compete with DAB™binding to TNFR1 (200 pg/ml) was approximately 70% of the maximum TNF-αresponse in this assay.

In order to further improve potency, single amino acid positions werediversified by oligo-directed mutagenesis at key positions suggested bythe error-prone lead consensus information. During this process animproved version of the DOM1h-131-8 clone, DOM1h-131-24 (originallynamed DOM1h-131-8-2 prior to correction) was isolated through BIACORE™screening that had a single K94R amino acid mutation (amino acidnumbering according to Kabat) and an RBA potency of 200-300 pM.

Further error-prone libraries based on this lead and the NNS libraryfrom which it was derived were generated and subjected to three roundsof phage selections using heat treatment (for method see Jespers L, etal., Aggregation-resistant domain antibodies selected on phage by heatdenaturation. Nat. Biotechnol. 2004 September; 22(9):1161-5). Duringthis selection, libraries were pooled and clones derived from round twoof the selection yielded DAB™s such as DOM1h-131-53 which wereconsidered to be more heat stable. It was hypothesised that these cloneswould possess better biophysical characteristics. Some frameworkmutations in clone DOM1h-131-53 were germlined to generate cloneDOM1h-131-83. This clone formed the basis for further diversificationvia oligo-directed individual CDR mutagenesis either using phage displayselection as described above or using the in-vitro compartmentalizationtechnology using emulsions. The phage display strategy generated leadsDOM1h-131-117 and DOM1h-131-151. The in-vitro compartmentalizationtechnology generated DOM1h-131-511.

At this stage these three leads were compared in biophysical andbiological assays and DOM1h-131-511 was the molecule with the bestproperties. Furthermore these molecules were tested for their resistanceto proteolytic cleavage in the presence of trypsin or leucozyme.Leucozyme consists of pooled sputum from patients with cystic fibrosisand contains high levels of elastase and other proteases and was used asa surrogate for in vivo conditions in lung diseases. This data indicatedthat all three leads DOM1h-131-117, DOM1h-131-151 and DOM1h-131-511 wererapidly degraded in presence of trypsin or leucozyme. This findingraised concerns about the in vivo persistence of DOM1h-131-511 when inthe patient and a strategy was developed to select for improvedresistance to trypsin. It was hypothesised that such improved trypsinresistance could have a beneficial effect on other biophysicalproperties of the molecule. Essentially the standard phage selectionmethod was modified to allow for selection in the presence of proteasesprior to selection on antigen. To this end a new phage vector wasengineered in which the c-myc tag was deleted to allow selections in thepresence of trypsin without cleaving the displayed DAB™ off the phage.DOM1h-131-511 based error-prone libraries were generated and cloned inthe pDOM33 vector (see FIG. 50 for pDOM33 vector map). Phage stocksgenerated from this library were pre-treated with either 1 mg/ml or 100μg/ml trypsin at 37° C. for 24 hours, subsequently protease inhibitorwhich was ROCHE COMPLETE PROTEASE INHIBITORS™ (2×) was added to blockthe trypsin activity prior to selection on the relevant antigen. Fourrounds of selection were performed. Soluble expressed TNFR1 bindingDAB™s were assessed using the BIACORE™ for their ability to bind TNFR1with or without the presence of proteases during one hour or overnightincubations at 37° C. in the presence or absence of trypsin (at 100μg/ml or 1000 μg/ml final trypsin concentration).

This led to the isolation of two lead molecules DOM1h-131-202 andDOM1h-131-206 which demonstrated improved protease resistance as shownby BIACORE™ antigen binding experiments. It is interesting to note thatDOM1h-131-202 contained only one mutation in CDR2 (V53D), all amino acidnumbering according to Kabat) in comparison to DOM1h-131-511, whereasDOM1h-131-206 contained only two mutations: the first mutation is thesame as in DOM1h-131-202 (V53D mutation in CDR2) and the second is aY91H mutation in FR3 (see FIG. 3). This Y91H mutation in FR3 does occurin the 3-20 human germline gene indicating that this residue occurs inhuman antibodies. The three clones DOM1h-131-511, DOM1h-131-202 andDOM1h-131-206 have amino acid sequences as shown in FIG. 3.

Activity of the Molecules was Determined as Below:

-   BIACORE™ binding affinity assessment of DOM1H-131-202, DOM1H-131-511    and DOM1H-131-206 for binding to human TNFR1.

The binding affinities of DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206for binding to human recombinant E. coli-expressed human TNFR1 wereassessed by BIACORE™ analysis. Analysis was carried out usingbiotinylated human TNFR1. 1400 RU of biotinylated TNFR1 was coated to astreptavidin (SA) chip. The surface was regenerated back to baselineusing mild acid elution conditions. DOM1H-131-202, DOM1H-131-511 andDOM1H-131-206 were passed over this surface at defined concentrationsusing a flow rate of 50 μl/min. The work was carried out on a BIACORE™3000 machine and data were analysed and fitted to the 1:1 model ofbinding. The binding data fitted well to the 1:1 model for all testedmolecules. All K_(D) values were calculated from k_(on) and k_(off)rates. BIACORE™ runs were carried out at 25° C.

The data below were produced from three independent experiments. In eachexperiment the results were calculated by averaging a number of fitsusing highest DAB™ concentrations for kd and lower concentrations forka. The data are presented as the mean and standard deviation (inbrackets) of the results (Table 1).

TABLE 1 BIACORE ™ data for DOM1H-131-202, DOM1H-131- 511 andDOM1H-131-206 binding to human TNFR1 k_(on) k_(off) K_(D) (nM)DOM1H-131-511 5.03E+05 5.06E−04 1.07 (511) (1.07E+05) (1.01E−04) (0.44)DOM1H-131-202 1.02E+06 5.42E−04 0.55 (202) (2.69E+05) (3.69E−05) (0.11)DOM1H-131-206 1.55E+06 7.25E−04 0.47 (206) (3.57E+05) (1.95E−04) (0.06)

DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 bound similarly and withhigh affinity to human TNFR1. DOM1H-131-202 and DOM1H-131-206 bind withaverage affinities of 0.55 nM and 0.47 nM respectively. BothDOM1H-131-202 and DOM1H-131-206 have a slightly better affinity incomparison to DOM1H-131-511 which has an average affinity of 1.07 nM.

Receptor Binding Assay:

The potency of the DAB™s was determined against human TNFR1 in areceptor binding assay. This assay measures the binding of TNF-alpha toTNFR1 and the ability of soluble DAB™ to block this interaction. TheTNFR1-FC fusion is captured on a bead pre-coated with goat anti-humanIgG (H&L). The receptor coated beads are incubated with TNF-alpha (10ng/ml), DAB™, biotin conjugated anti-TNF-alpha and streptavidin ALEXAFLUOR™ 647 in a black sided clear bottomed 384 well plate. After 6 hoursthe plate is read on the ABI 8200™ cellular detection system and beadassociated fluorescence determined. If the DAB™ blocks TNF-alpha bindingto TNFR1 the fluorescent intensity will be reduced.

Data was analysed using the ABI 8200™ analysis software. Concentrationeffect curves and potency (EC₅₀) values were determined using GRAPHPADPRISM™ and a sigmoidal dose response curve with variable slope. Theassay was repeated on three separate occasions. A TNF-alpha dose curvewas included in each experiment (FIGS. 38 and 39). The concentration ofTNF-alpha used to compete with DAB™ binding to TNFR1 (10 ng/ml) isapproximately 90% of the maximum TNF-alpha response in this assay.

A representative graph is shown in FIG. 39 showing the ability of DAB™sto inhibit the binding of TNF-alpha to TNFR1. In all three experimentsthe negative control samples (HEL4, an anti-hen egg white lysozyme DAB™and V_(H) dummy) weakly inhibit the interaction between TNF-alpha andTNFR1 at high concentrations. The average potency (EC₅₀) values for thetest samples and positive controls (anti-TNFR1 mAb obtained from R&DSystems, mAb225) and ENBREL™ (etanercept; a dimeric fusion consisting ofTNFR2 linked to the Fc portion of IgG1; licensed for the treatment ofrheumatoid arthritis) are shown in Table 2.

TABLE 2 Potency (EC₅₀) values for DOM1H-131-202, DOM1H-131-206 andDOM1H-131-511 in a TNFR1 receptor binding assay for three repeatexperiments. Sample Average EC₅₀ (nM) SEM DOM1H-131-202 0.11 0.008DOM1H-131-206 0.07 0.01 DOM1H-131-511 0.19 0.01 ENBREL ™ (Etanercept)0.20 0.07 Anti-TNFR1 mAb # mAb225 0.08 0.003

In this assay DOM1H-131-206 appears more potent than the other two DAB™sbeing tested and has a similar potency to the commercially availableanti-TNFR1 mAb, MAB225 (R and D Systems).

Expression of lead clones from Pichia pastoris was carried out asdescribed below: The primary amino acid sequence of the three leadmolecules was used to produce codon optimised genes for secretedexpression in Pichia pastoris. There is 75% sequence identity betweenthe codon optimized and the non-codon optimized DOM1H-131-206. The threesynthetic genes were cloned into the expression vector pPIC-Zα (fromInvitrogen) and then transformed into two Pichia strains, X33 and KM71H.The transformed cells were plated out onto increasing concentrations ofZEOCIN™ (100, 300, 600 and 900 μg/ml) to select for clones with multipleintegrants. Approximately 15 clones for each cell line and constructwere selected for expression screening. As the correlation betweenhigh/low gene copy number and expression level is not fully understoodin Pichia pastoris, several clones were picked from across the ZEOCIN™concentration range. 5 L fermenter runs were carried out using clonesthat had not been extensively screened for high productivity. Thisallowed the production of significant amounts of material for furtherstudies.Material Production for Protein Characterisation:

Protein A based chromatography resins have been extensively used topurify V_(H) DAB™s from microbial culture supernatants. Although thisallows a single step purification method for producing high puritymaterial, usually >90% in most cases, for some molecules the low pHelution conditions can result in the formation of aggregates. There isalso the issue of the limited capacity of affinity resins for DAB™s;this would mean the use of significant quantities of resin to processfrom fermenters. In order to produce high quality material forcharacterisation and further stability and nebuliser studies, adownstream purification process was devised using a mixed modal chargeinduction resin as the primary capture step followed by anion exchange.Without significant optimisation, this allowed the recovery of ˜70% ofthe expressed DAB™ at a purity of ˜95%.

For the capture step on the mixed modal charge induction resin, CAPTO™MMC from GE Healthcare, column equilibration is performed using 50 mMsodium phosphate pH6.0 and the supernatant is loaded without any needfor dilution or pH adjustment. After washing the column, the protein iseluted by pH gradient using an elution buffer which is 50 mM Tris pH9.0. The specific wash and gradient conditions will vary slightlydepending on the pI of the protein being eluted.

The eluate peak is then further purified with a flow through step usinganion exchange chromatography. This removes residual HMW contaminationsuch as alcohol oxidase and reduces endotoxin. The resin is equilibratedwith either PBS or phosphate buffer pH 7.4 without salt. Upon loadingthe eluate from CAPTO™ MMC onto the anion exchange resin the DAB™ doesnot bind and is recovered from the flow through. Endotoxin and othercontaminants bind to the resin. The presence of salt if using PBS bufferimproves protein recovery to 91% for this step rather than 86% recoveryachieved without salt. However the presence of salt reduces theeffectiveness of endotoxin removal such that a typical endotoxin levelof DAB™ following this step with the inclusion of salt was measured as58 EU/ml compared with a level of <1.0 EU/ml obtained when no salt waspresent.

Protein Characterisation:

The material produced from the 5 L fermenter runs was characterised foridentity using electrospray mass spectrometry, amino terminal sequencingand isoelectric focusing and for purity using SDS-PAGE, SEC and GELCODE™glycoprotein staining kit (Pierce).

Identity:

The amino terminal sequence analysis of the first five residues of eachprotein, was as expected (EVQLL . . . ) (SEQ ID NO: 259). Massspectrometry was performed on samples of the proteins which had beenbuffer exchanged into 50:50 H₂O:acetonitrile containing 0.1% glacialacetic acid using C4 ZIP-TIPS™ (Millipore). The measured mass for eachof the three proteins was within 0.5 Da of the theoretical mass based onthe primary amino acid sequence (calculated using average masses) whenallowing for a mass difference of −2 from the formation of the internaldisulphide bond. IEF was used to identify the proteins based on their pIwhich was different for each protein.

Purity:

The three proteins were loaded onto non-reducing SDS-PAGE gels in 1 μgand 10 μg amounts in duplicate. A single band was observed in allinstances. Size exclusion chromatography was also performed todemonstrate levels of purity. For size exclusion chromatography (SEC)100 μg of each protein were loaded onto a TOSOH G2000 SWXL™ columnflowing at 0.5 ml/min. Mobile phase was PBS/10% ethanol.

Investigation of DAB™ Stability for Candidate Selection:

For the indication of COPD, it would be necessary to deliver the DAB™into the lung, e.g. using a nebuliser device. This would mean theprotein could potentially experience a range of shear and thermalstresses depending on the type of nebuliser used and could be subjectedto enzymatic degradation by proteases in the lung environment. It wasdetermined if the protein could be delivered using this type of device,form the correct particle size distribution and remain functionalfollowing nebuliser delivery. Therefore the intrinsic stability of eachmolecule to a range of physical stresses was investigated to determinethe baseline stability and the most sensitive stability indicatingassays. As the stability of each protein will be dependent on the buffersolution it is solubilised in, some pre-formulation work was necessary.This information, such as buffer, pH, would also be useful forunderstanding the stability of the protein during the downstreampurification process and subsequent storage. In order to characterisethe changes in the molecules during exposure to a range of physicalstresses, a range of analytical techniques were used such as sizeexclusion chromatography (SEC), SDS-PAGE and isoelectric focusing (IEF).

Assessment of Protease Stability of DOM1H-131-202, DOM1H-131-511 andDOM1H-131-206:

The protease stability of DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206was assessed by BIACORE™ analysis of the residual binding activity afterpre-incubation for defined timepoints in excess of proteases.Approximately 1400 RU of biotinylated TNFR1 was coated to a streptavidin(SA) chip. 250 nM of DOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 wasincubated with PBS only or with 100 μg/ml of trypsin, elastase orleucozyme for 1, 3, and 24 hours at 30° C. The reaction was stopped bythe addition of a cocktail of protease inhibitors. The DAB™/proteasemixtures were then passed over the TNFR1 coated chip using referencecell subtraction. The chip surface was regenerated with 10 ul 0.1Mglycine pH 2.2 between each injection cycle. The fraction ofDOM1H-131-202, DOM1H-131-511 and DOM1H-131-206 bound to human TNFR1 (at10 secs) pre-incubated with proteases was determined relative to DAB™binding without proteases. BIACORE™ runs were carried out at 25° C.

The data was produced from three independent experiments. The bar graphindicates mean values and the error bars indicate standard deviation ofthe results (for results see FIG. 24).

It was found that DOM1H-131-202 and DOM1H-131-206 were shown to havegreater resistance to proteolytic degradation by trypsin, elastase orleucozyme in comparison to DOM1H-131-511. The difference betweenDOM1H-131-202 and DOM1H-131-206 in comparison to DOM1H-131-511 is mostpronounced after 1 hr with trypsin and after 3 hrs with elastase orleucozyme.

Thermal Stability as Determined Using DSC:

In order to determine at which pH the molecules had the greateststability, differential scanning calorimeter (DSC) was used to measurethe melting temperatures (T_(m)) of each DAB™ in Britton-Robinsonbuffer. As Britton-Robinson is made up of three component buffer systems(acetate, phosphate and borate), it is possible to produce a pH rangefrom 3-10 in the same solution. The theoretical pI was determined fromthe proteins primary amino acid sequence. From the DSC, the pH at whichthe DAB™s had their greatest intrinsic thermal stability was found to bepH 7 for DOM1H-131-202 (202), pH 7-7.5 for DOM1H-131-206 (206) and pH7.5 for DOM1H-131-511 (511). For all subsequent stress and stabilitywork the following pHs were used for each DAB™; for DOM1H-131-202 (202)and DOM1H-131-206 (206) pH 7.0 and for DOM1H-131-511 (511) pH 7.5 inBritton-Robinson buffer. The results are summarised in Table 3 below:

TABLE 3 Summary of the pH and T_(m)s of DOM1H-131-202 (202),DOM1H-131-206 (206) and DOM1H-131-511 (511) as determined by DSC inBritton-Robinson buffer at 1 mg/ml pH that gives Tm (° C.) of greatestintrinsic the DAB ™ at DAB ™ thermal stability the given pHDOM1H-131-202 (202) 7.0 68.6 DOM1H-131-206 (206) 7.0-7.5 65.8DOM1H-131-511 (511) 7.5 58.0Intrinsic Solubility Testing:

All the lead DAB™s were concentrated in centrifugal VIVASPIN™concentrators (5K cut-off), to determine their maximum solubility andthe levels of recovery upon concentration. Experiments were performed inBritton-Robinson buffer at the most stable pH. Sample volumes andconcentrations were measured over a time course and deviation fromexpected concentration recorded as well as percent recovery of thesample.

It was found that all proteins could be concentrated to over 100 mg/mlin Britton-Robinson buffer. Both DOM1H-131-202 (202) and DOM1H-131-206(206) showed lower recoveries than expected compared to DOM1H-131-511(511), but still within acceptable levels.

Nebuliser Delivery of the Lead DAB™s:

By testing different nebulisers and formulation buffers it wasdemonstrated that the DAB™ could effectively be delivered using a widerange of nebulising devices. More importantly, it was shown for thefirst time that nebulisation of the DAB™ in the formulation bufferproduced the desired particle size distribution (compared using thepercentage of droplets <5 μm) for effective lung delivery whilstmaintaining protein functionality. This is further described below.

Comparison of Performance in Various Devices:

DOM1H-131-511 (511) was tested in six nebuliser devices comprising twodevices from each of the three main groups of nebulisers for liquidformulations i.e. ultrasonic nebulisers, jet nebulisers and vibratingmesh nebulisers. In each device the DAB™ was tested at 5 mg/ml with arange of PEG concentrations. For each sample the percentage of dropletsize <5 μm was measured using a Malvern SPRAYTEC™ Device (MalvernInstruments Limited, UK) and the results are shown in FIG. 35. Thestability of each sample after being nebulised was assessed using SEC toanalyse the amount of sample which had dimerised both in the materialremaining in the cup and in collected aerosol. The results may be seenin FIG. 36. The less the extent of dimer formation the greater thestability.

Most devices can deliver 40% or more of the liquid formulation in thecorrect size range but the EFLOW™ (a vibrating mesh nebuliser device)and PARI LC+™ (a jet nebuliser) devices perform better, with the PARILC*™ (star) device delivering more than 80% when PEG is included in thebuffer. This increase in delivery with PEG is also observed with theEFLOW™ and, to a lesser extent, with the PARI LC+™.

Importantly Activity of the DAB™ was Also Found to be Retained afterNebulisation (See Results in FIG. 8)

Effect of Buffer Additives:

Due to the lower stability of DOM1H-131-511 (511), the 50 mM phosphateformulation buffer contained both PEG 1000 and sucrose (and has aviscosity which is within the range which is defined as about equal tothe viscosity of a solution of about 2% to about 10% PEG 1000 in 50 mMphosphate buffer containing 1.2% (w/v sucrose) to help protect the DAB™from both shear and thermal stress. As both DOM1H-131-202 (202) andDOM1H-131-206 (206) have higher T_(m)'s and showed considerably improvedstability to thermal stress, all the molecules were tested in both theoriginal formulation buffer and in Britton-Robinson buffer (which has alower viscosity than the formulation buffer). The DAB™s were tested inboth the EFLOW™ and PARI LC+™ devices with run time of 3.5 minutes at aprotein concentration of 5 mg/ml and the particle size distributiondetermined using a Malvern SPRAYTEC™ Device. As a comparison, a marketeddrug for cystic fiborosis (designated standard protein X) that isdelivered using a nebuliser device, was tested in its own formulationbuffer. The results are shown in FIG. 37. For good delivery anddistribution into the deep lung, the ideal particle size is less than 6microns, e.g. <5 μm. All the DAB™s give comparable levels of particlesizes that were less than 5 μm in both the Britton-Robinson buffer andformulation buffer (as described earlier). However, the higher viscosityof the formulation buffer could be particularly beneficial for producingparticles within the correct size range, e.g. particles <5 μm.

The concentration of the DAB™ in the cup of the device was determined byA₂₈₀ measurements before and after nebulisation. It was found that theprotein concentration did not change significantly indicating thatneither the protein nor vehicle is preferentially nebulised duringdelivery.

CONCLUSION

It has been demonstrated as described above that polypeptides such asDAB™s can be nebulised in a range of commercially available nebuliserdevices and importantly that they retain stability and biologicalactivity after nebulisation and there is no significant aggregationobserved following nebulisation. When viscosity enhancing excipients,such as PEG are added to the buffer formulation, particle sizedistribution and percentage droplet size less than 5 μm can be improved,thus potentially improving DAB™ delivery to the deep lung.

Delivery of DAB™ to the lung can also be improved by increasing the DAB™concentration for example a concentration of up to about 40 mg/ml anddelivery time without any reduction in DAB™ stability or activity.

Example 1

Phage Vector pDOM13

A filamentous phage (fd) display vector, pDOM13 was used. This vectorproduces fusion proteins with phage coat protein III. The multiplecloning site of pDOM13 is illustrated in FIG. 1. The genes encodingDAB™s were cloned as SalI/NotI fragments.

Example 2

Test Protease Selections on Phage-Displayed Domain Antibodies (DAB™s)with a Range of Resistance to Trypsin

The genes encoding DAB™s DOM4-130-54 which binds IL-1R1, DOM1h-131-511which binds TNFR1, and DOM15-10, DOM15-26 and DOM15-26-501, which bindVEGFA, were cloned in pDOM13 and phages displaying these DAB™s wereproduced according to standard techniques. Phages were purified by PEGprecipitation, resuspended in PBS and titered.

The above DAB™s displayed a range of ability to resist degradation bytrypsin when tested as isolated proteins. Resistance to degradation wasassessed as follows: DAB™ (1 mg/ml) in PBS was incubated with trypsin at40 μg/ml at 30° C., resulting in a molecular ratio of 25:1 DAB™:trypsin.Samples (30 μl) were taken immediately before addition of trypsin, andthen at T=1 hour, 3 hours, and 24 hours. Protease activity wasneutralized by addition of ROCHE COMPLETE PROTEASE INHIBITORS™ (2×)followed by immersion in liquid nitrogen and storage on dry ice. 15 μgof each DAB™ sample was subsequently analyzed by electrophoresis on aNOVEX™ 10-20% Tricine gel and proteins were stained with SUREBLUE™ (1×).

Both DOM15-10 and DOM15-26-501 were significantly digested during thefirst three hours. DOM15-26, DOM4-130-54 and DOM1h-131-511 were morestable, with digestion of the DAB™s only becoming apparent after 24hours (FIG. 2).

The phage-displayed DAB™s were also incubated in the presence of trypsinto evaluate if trypsin resistance of phage-displayed DAB™s correlatedwith the results obtained with the isolated soluble DAB™s. Variousconcentrations of trypsin and incubation times were tested. In allcases, after neutralization of trypsin with ROCHE COMPLETE PROTEASEINHIBITORS™, the phages were tested for their ability to bind a genericligand: protein A, which binds all V_(H) domain antibodies (e.g.,DOM1h-131, DOM15-26, DOM15-26-501) or protein L, which binds all V_(K)domain antibodies (e.g., DOM4-130-54, DOM15-10). Phage were also testedfor binding to target antigens. In both cases, binding was assumed tocorrelate with retention of the DAB™ structural integrity throughresistance to proteolysis. The binding activity was measured either byELISA (using conjugated antibodies against phage) or by elution of boundphages and titre analysis following infection of exponentially growingE. coli TG1 cells.

Tests with DOM15-10, DOM15-26 and DOM15-26-501 on Phage

Each DAB™ was treated for one hour at room temperature with a range oftrypsin concentrations (100 μg/ml, 10 μg/ml and 0 μg/ml). Trypsinactivity was blocked with ROCHE COMPLETE PROTEASE INHIBITORS™ (1×) andthen the phages were diluted in 2% MARVELL™ in PBS, incubated with 50 nMof biotinylated antigen (recombinant human VEGF (R&D systems)) for onehour at room temperature. Strepavidin-coated beads (DYNABEADS™ M-280(Invitrogen)) that were pre-blocked for one hour at room temperaturewith 2% MARVELL™ in PBS were added, and the mixture was then incubatedfor five minutes at room temperature. All of the incubation steps withDYNABEADS™ were carried out on a rotating wheel. Unbound phages werewashed away by washing the beads eight times with 1 ml of 0.1% TWEEN-20™in PBS. Bound phages were eluted with 0.5 ml of 0.1M Glycine pH2.2 andneutralized with 100 μl of 1M Tris-HCL pH 8.0. Eluted phage were used toinfect exponentially growing TG1 cells (one hour at 37° C.) and platedon tetracycline plates. Plates were incubated overnight at 37° C. andcolony counts were made (see Table 4). The best results were observedfrom selection with incubation with 100 μg/ml trypsin. There was about a10-fold increase in the yield of DOM15-26 in comparison to DOM15-10 andDOM15-26-501.

A second experiment was done to further confirm these results under moresevere incubation conditions. Phage displayed DAB™s were treated for 1hour or 2 hours at 37° C. with agitation (250 rpm). The best resultswere observed from selections with 2 hour incubation with 100 ug/mltrypsin. The yield of DOM15-26 was 200-fold higher than the yield ofDOM15-26-501 and 1000-fold higher than the yield of DOM15-10.

In a third experiment, phages displaying DOM15-26 and DOM15-26-501 weremixed 1:1 at the start. They were then either incubated with trypsin(1000 μg/ml) or without trypsin for two hours at 37° C. with agitation(250 rpm), and then selected for antigen binding as described above.Sequencing of ten colonies from each selection revealed a mixedpopulation of clones for selection without trypsin pre-treatment(DOM15-26: 4/10; DOM15-26-501: 6/10), whereas all clones from theselection with trypsin encoded for DOM15-26 as expected.

These experiments indicate that a selection pressure can be obtained byadding a protease to phages displaying DAB™s, such that phagesdisplaying the most proteolytically stable DAB™s are preferentiallyselected (following panning on a generic ligand or the antigen).

TABLE 4 1:1 Length of Trypsin DOM15-26 DOM15-26-501 mixed DOM15-10Experiment incubation Temp. concentration titre titre titre titre 1 1 hrRoom 100 μg/ml 1.6 × 10⁸ 6.3 × 10⁷ 1.1 × 10⁷ input 10¹⁰ temp 1 hr Room 10 μg/ml  3 × 10⁸ 4.4 × 10⁸ 2.4 × 10⁸ temp 1 hr Room  0 μg/ml 0.9 × 10⁸ 2 × 10⁸ 0.7 × 10⁸ temp 2 1 hr, 250 rpm 37° C. 100 μg/ml  2 × 10⁷  1 ×10⁶  1 × 10⁵ input 10⁹  2 hr, 250 rpm 37° C. 100 μg/ml  1 × 10⁷  6 × 10⁴ 1 × 10⁴ 2 hr, 250 rpm 37° C.  0 μg/ml 5.4 × 10⁷ 4.1 × 10⁷  3 × 10⁸ 3  2h, 250 rpm 37° C. 100 μg/ml 2.3 × 10⁸  8 × 10⁵ 6.8 × 10⁷ input 10¹⁰  2h, 250 rpm 37° C.  0 μg/ml 3.9 × 10⁸ 4.4 × 10⁸ 4.8 × 10⁸

Tests with DOM4-130-54 on Phage

DOM4-130-54 was tested in a similar protocol as described above. Theparameters that were varied were: concentration of trypsin, temperatureand length of incubation. Biopanning was done against IL-RI-Fc (a fusionof IL-1RI and Fc) at 1 nM concentration in PBS. Significant reductionsin phage titre were only observed after incubation of the phage with 100μg/ml trypsin overnight at 37° C. (see Table 5).

TABLE 5 Length of Trypsin incubation Temperature concentration Titre 1hr Room temp 100 μg/ml   1.8 × 10¹⁰ 1 hr Room temp 10 μg/ml 7.2 × 10⁹ 1hr Room temp  0 μg/ml 6.6 × 10⁹ Overnight Room temp 100 μg/ml  2.16 ×10⁹  Overnight Room temp 10 μg/ml 7.2 × 10⁹ Overnight Room temp  0 μg/ml7.8 × 10⁹ Overnight 37° C. 100 μg/ml  2.04 × 10⁶  Overnight 37° C. 10μg/ml 3.84 × 10⁸  Overnight 37° C.  0 μg/ml 7.2 × 10⁹

Tests with DOM1h-131 Phage

DOM1h-131 phage (closely related to DOM1h-131-511 by amino acidsequence) were treated with 0 μg/ml, 10 μg/ml, 100 μg/ml and 1000 μg/mltrypsin for one hour at room temperature. Digestion was inhibited by theaddition of 25× ROCHE COMPLETE PROTEASE INHIBITORS™. Serial 2-folddilutions of the phage were carried out down an ELISA plate coated with1 nM TNFRI, and binding phage were detected with anti-M13-HRP. Theresults are shown below in Table 6.

TABLE 6 DOM1h-131 Trypsin concentration 1 100 10 0 Phage mg/ml μg/mlμg/ml μg/ml input 0.284 0.418 0.784 0.916 4.51E+10 0.229 0.377 0.8020.944 2.26E+10 0.183 0.284 0.860 0.949 1.13E+10 0.133 0.196 0.695 0.9625.64E+09 0.114 0.141 0.573 0.946 2.82E+09 0.089 0.115 0.409 0.8501.41E+09 0.084 0.084 0.286 0.705 7.05E+08 0.080 0.084 0.213 0.5773.52E+08

These test experiments clearly show that 100 μg/ml of trypsin and atemperature of 37° C. are appropriate to apply a selection pressure onphages displaying DAB™s of various degrees of resistance to proteolysisby trypsin. Incubation time with the protease can be optimized for eachphage-displayed DAB™, if desired.

Example 3

Protease Selection of Phage-Displayed Repertoires of Domain Antibodies

Four repertoires were created using the following DAB™s as parentmolecules: DOM4-130-54, DOM1h-131-511, DOM15-10 and DOM15-26-555. Randommutations were introduced in the genes by PCR using the STRATAGENEMUTAZYME II™ kit, biotinylated primers and 5-50 pg of template for a 50μl reaction. After digestion with SalI and NotI, the inserts werepurified from undigested products with streptavidin-coated beads andligated into pDOM13 at the corresponding sites. E. coli TB1 cells weretransformed with the purified ligation mix resulting in largerepertoires of tetracycline-resistant clones: 8.5×10⁸ (DOM4-130-54),1.5×10⁹ (DOM1h-131-511), 6×10⁸ (DOM15-10) and 3×10⁹ (DOM15-26-555).

Phage libraries were prepared by double precipitation with PEG andresuspended in PBS.

The rates of amino acid mutations were 2.3 and 4.4 for the DOM1h-131-511and DOM4-130-54 repertoires, respectively. The functionality wasassessed by testing 96 clones in phage ELISA using wells coated withprotein A or protein L (at 1 μg/ml). 62.5% and 27% of the clonesexhibited functional display of DAB™s in the DOM1h-131-511 andDOM4-130-54 repertoires, respectively.

The rates of amino acid mutations were 2.5 and 4.6 for the DOM15-10 andDOM15-26-555 repertoires, respectively. The functionality was assessedby testing 96 clones in phage ELISA using wells coated with protein A orprotein L (at 1 μg/ml). 31.3% and 10.4% of the clones exhibitedfunctional display of DAB™s in the DOM15-10 and DOM15-26-555repertoires, respectively.

DOM4-130-54 and DOM1h-131-511 Repertoires

Four rounds of selection were carried out with these libraries to selectfor DAB™s with improved protease resistance.

The first round of selection was by antigen binding (1 nM or 10 nMantigen) without protease treatment to clean-up the library to removeany clones that no longer bound antigen with high affinity. The outputsfrom round 1 were in the 10⁸-10¹⁰ range (compared to an input of 10¹¹phage) indicating that the majority of the library bound antigen withhigh affinity.

In round 2, protease treatment with 100 μg/ml trypsin was introduced,and the outputs are as shown below in Table 7:

TABLE 7 Trypsin incubation DOM1h-131-511 DOM4-130-54 conditions librarylibrary 37° C. overnight 1.86 × 10⁶  2.1 × 10⁶ 37° C. 2 hrs 4.8 × 10⁸5.1 × 10⁸ Room temperature 2 hrs 1.2 × 10⁹ 4.62 × 10⁹  No trypsin  ~1 ×10⁹  ~4 × 10⁹ No antigen 1.8 × 10⁴  <6 × 10³

There was significant selection when the DAB™s were treated with trypsinat 37° C. overnight. This output was taken forward to round 3, where thephage were treated with either 1 mg/ml or 100 μg/ml trypsin at 37° C.for 24 hours. The titres of the trypsin treated phage from round 3 were10⁵-10⁶ for the DOM1h-131-511 repertoire and 10⁷-10⁸ for theDOM4-130-154 repertoire.

All outputs from round 3 (DOM1h-131-511 and DOM4-130-154 with 1 mg/mland 100 μg/ml) underwent a fourth round of selection against 1 nMantigen with 100 μg/ml trypsin. The titres were in the range of 10⁶-10⁸,similar to that seen in round 3. Some enrichment was seen for theDOM1h-131-511 repertoire, but no enrichment was seen for the DOM4-130-54repertoire.

DOM15-10 and DOM15-26-555 Repertoires

The first round of selection was carried out with 2 nM biotinylatedhVEGF (human vascular endothelial growth factor) concentration andwithout protease treatment to clean-up the library to remove any clonesthat no longer bound antigen with high affinity. The outputs from round1 were about 10⁸ (compared to an input of 10¹⁰ phage for DOM15-10 and10¹¹ phage for DOM15-26-555) indicating that the majority of the librarybound antigen with high affinity.

The second and third rounds of selection were performed with 2 nMbiotinylated hVEGF. Prior to panning on hVEGF, the phages were incubatedin the presence of trypsin (100 μg/ml) at 37° C. in a shaker (250 rpm).Incubation time was one hour for the DOM15-10 repertoire and two hoursfor the DOM15-26-555 repertoire.

The outputs were as follows: 1.5×10⁶ and 9×10⁵ for the second and thirdrounds of selection with the DOM15-10 repertoire; 2.2×10⁸ and 3.9×10⁹for the second and third rounds of selection with the DOM15-26-555.

Example 4

Analysis of Selection Outputs: DOM4-130-54 and DOM1h-131-511 Repertoires

All outputs from round 3 and round 4 were subcloned into the pDOM5vector and transformed into JM83 cells. The pDOM5 vector is apUC119-based vector. Expression of proteins is driven by the Placpromoter. A GAS1 leader sequence (see WO 2005/093074) ensured secretionof isolated, soluble DAB™s into the periplasm and culture supernatant ofE. coli JM83. 96 and 72 individual colonies from round 3 and round 4were randomly picked for expression

12-24 clones were sequenced from each round 3 and round 4 output.Consensus mutations were observed in both selections and approximately25 clones harboring consensus motifs were chosen for furthercharacterization. The amino acid sequences of these clones are shown inFIG. 3 (DOM1h-131-511 selected variants) and FIG. 4 (DOM4-130-54selected variants) and listed as DNA sequences in FIGS. 19A-19L. Theamino acids that differ from the parent sequence in selected clones arehighlighted (those that are identical are marked by dots). The loopscorresponding to CDR1, CDR2 and CDR3 are outlined with boxes.

These clones were expressed in a larger amount, purified on protein L(for DOM4-130-54 variants) and protein A (for DOM1h-131-511 variants)and tested for antigen binding on BIACORE™ after one hour or overnightincubation at 37° C. in the presence or absence of trypsin (100 μg/ml or1000 μg/ml final concentration).

Generally, the outputs from the DOM4-130-54 selections were more stablewith most clones remaining resistant to trypsin for one hour and thebest clones resistant overnight. In comparison, a small number of clonesfrom the DOM1h-131-511 selections were resistant to trypsin for onehour, whilst none of the clones were resistant overnight.

Example 5

Analysis of Selection Outputs: DOM15-10 and DOM15-26-555 Repertoires

The effectiveness of selection with trypsin pre-treatment was firsttested on monoclonal phage ELISA with and without trypsin digestion.Eighteen colonies from the second round of selection and 24 coloniesfrom the third round of selection of each library were picked. ClonesDOM15-10, DOM15-26-501 and DOM15-26 were used as controls. Additionalcontrols included amplified and purified phage solution from eachlibrary after second and third rounds of trypsin selection.

Each phage sample was divided into two fractions, the first was treatedwith 100 ug/ml trypsin, the second was not treated with trypsin.Incubation of both fractions was carried out for one hour at 37° C. withagitation (250 rpm) and blocked by adding ROCHE COMPLETE PROTEASEINHIBITORS™ (1×).

Phage ELISA was performed using the trypsin-digested and undigestedsamples. ELISA wells were coated with NEUTRAVIDIN™ in 0.1M bicarbonatebuffer at a concentration of 1 μg/ml. After the washing steps with PBSand blocking of the antigen-coated wells with 1% TWEEN-20™ in PBS forone hour at room temperature, the wells were coated with biotinylatedhVEGF diluted in 1% TWEEN-20™ in PBS at a concentration of 100 ng/ml.Next, the wells were washed with PBS and treated or untreated phagesupernatants diluted 1:1 with 1% TWEEN-20™/PBS, were added. After 30minutes of incubation at 37° C., the wells were washed with 1%TWEEN-20™/PBS, followed by a 30 minute incubation at 37° C. withanti-M13 phage-HRP conjugate (diluted 1/5000 in 1% TWEEN-20™/PBS). Thewells were then washed with PBS and peroxidase. Reaction was initiatedby adding SUREBLUE™ reagent. After about ten minutes, the reaction wasstopped with an equivalent volume of 1M HCl and the wells were read atOD_(450 nM).

ELISA read-outs of unstable controls DOM15-10 and DOM15-26-501 treatedwith trypsin gave an OD₄₅₀ lower than 0.404 and this value was assumedas a border value of an unstable clone. All samples that gave an ODlower than 0.404 were considered to be unstable. All samples above thatvalue were considered to be stable.

TABLE 8 Trypsin No trypsin 2nd 3rd 2nd 3rd Library selection selectionselection selection DOM15-10   33%  89% 100% 100% DOM15-26-555 94.4%100% 100% 100%

Table 8 shows the percentage of stable clones after the second and thirdrounds of trypsin selection of each library. The enrichment of trypsinresistant clones is visible in both libraries after the third round ofselection. The values of control ELISA wells containing amplifiedpurified phage mix after each selection were much higher than 0.404 ineach case after trypsin digestion. Moreover, a small increase in signalwas observed when comparing trypsin-treated phage from the third roundof selection with trypsin-treated phage from the second round ofselection. The DOM15-10 phage library showed an increase of about 14% ofthe starting value. DOM15-26-555 phage library showed an increase thatrepresents about 2% of the starting value.

Overall these results show that selection with trypsin pre-treatment waseffective to select trypsin-resistant phage clones from the DOM15-10 andDOM15-26-555 repertoires.

All outputs from the second and third rounds of selection (DOM15-26-555)and from the third round of selection only (DOM15-10) were subclonedinto the pDOM5 vector and transformed into HB2151 electrocompetentcells. The pDOM5 vector is a pUC119-based vector. Expression of proteinsis driven by the Plac promoter. A GAS1 leader sequence ensured secretionof isolated, soluble DAB™s into the periplasm and culture supernatant ofE. coli HB2151. 184 individual colonies from each round of selection (3and 4) were randomly picked for expression in 1 ml culture volumes.

Bacterial supernatants were diluted in HBS-EP BIACORE™ buffer (1:1volume ratio) and split to duplicates. Trypsin was added to only onevial at a final concentration of 20 μg/ml. Incubation was carried outfor 40 minutes at 37° C. with agitation (250 rpm). After blocking thereaction with ROCHE COMPLETE PROTEASE INHIBITORS™ (1×), both trypsintreated and untreated phage supernatants were tested on BIACORE™ 3000for antigen binding (2,000 RU of biotinylated hVEGF on a SA sensorchip).

The criteria for picking clones were: a decrease in antigen binding of<15% of DAB™s treated with trypsin relative to untreated DAB™s (based onmax RU reached on selected time point), which would reflect DAB™sstability to protease treatment in general; and off-rate decrease of<40% between two time points during dissociation of a DAB™ from theantigen. Based on these values, 60 clones from both the second and thirdrounds of selection of the DOM15-26-555 library and 17 clones from thethird round of selection of the DOM15-10 library were sequenced.Consensus mutations were observed in both libraries' outputs and 17clones from each library harboring consensus motifs were chosen forfurther characterization. The amino acid sequences of these clones areshown in FIG. 5 (DOM15-26-555 selected variants) and FIG. 6 (DOM15-10selected variants) and listed as DNA sequences in FIGS. 20A-20E. Theamino acids that differ from the parent sequence in selected clones arehighlighted (those that are identical are marked by dots). The loopscorresponding to CDR1, CDR2 and CDR3 are outlined by boxes.

These clones were expressed in 50 ml expression cultures, purified onprotein A (for DOM15-26-555 variants) or protein L (for DOM15-10variants) diluted to 100 nM concentration in HBS-EP buffer and testedfor antigen binding on BIACORE™ after 1.5 hours of incubation at 37° C.with agitation (250 rpm) in the presence or absence of trypsin (20 μg/mlfinal concentration).

These clones were also tested for trypsin resistance using the methoddescribed in Example 2. Proteins were buffer exchanged to PBS andconcentrated to 1 mg/ml. 25 μg of protein was mixed with 1 μg of trypsin(Promega) and incubated for 0 hours and 24 hours at 30° C. After thistime, the reaction was blocked with ROCHE COMPLETE PROTEASE INHIBITORS™(1×) and DTT, as well as loading agent, was added Samples were denaturedfor five minutes at 100° C. Then 15 μg of each sample was analyzed byelectrophoresis on NOVEX™ 10-20% Tricine gels and proteins were stainedwith SUREBLUE™ (1×).

Generally, the outputs from the DOM15-26-555 selections were morestable, with most clones remaining resistant to trypsin for 1.5 hourswhen tested on BIACORE™ and overnight when run on SDS-PAGE. Incomparison, only a small number of clones from the DOM15-10 selectionswere resistant to trypsin for overnight treatment when run on SDS-PAGE.

Example 6

Identification of DOM1h-131-511 Variants

DOM1h-131-203, DOM1h-131-204 and DOM1h-131-206 were analyzed in furtherdetail. They were compared on the BIACORE™ at a DAB™ concentration of500 nM after incubation with different concentrations of trypsin(ranging from 0 to 100 μg/ml) overnight at 37° C. The BIACORE™ tracesare shown in FIG. 7. The results clearly show that both variants aremore resistant than their parent to proteolysis at high concentration oftrypsin (100 μg/ml). Two of the DAB™s, DOM1h-131-202 and DOM1h-131-206,were also compared along with their parent against a range of otherproteases including leucozyme, elastase and pancreatin under theconditions described above, with a protease concentration of 100 μg/ml.The DAB™s showed increased resistance to proteolysis compared to theparent against all proteases tested. The BIACORE™ traces for elastaseand leucozyme are shown in FIG. 8.

5 μM of each DAB™ was treated with 100 μg/ml sequencing grade trypsinfor 0, 1, 3 and 24 hours. The reaction was inhibited with 25× ROCHECOMPLETE PROTEASE INHIBITOR™ and the reactions were run on a 4-12%NOVEX™ Bis-Tris gel. The gels are shown in FIG. 9.

Example 7

Identification of DOM4-130-54 Variants

DOM4-130-201 and DOM4-130-202 were analyzed in further detail. They werecompared on the BIACORE™ at a DAB™ concentration of 500 nM afterincubation with different concentrations of trypsin (ranging from 0 to100 μg/ml) overnight at 37° C. The BIACORE™ traces are shown in FIG. 10.The results clearly show that all three variants are more resistant thantheir parent to proteolysis at high concentrations of trypsin (100μg/ml). DOM4-130-201 and DOM4-130-202 were also compared with the parentagainst a range of other proteases including leucozyme, elastase andpancreatin under the conditions described above with a proteaseconcentration of 100 μg/ml. Although the results were less apparent thanwith trypsin, the lead DAB™s showed increased resistance to proteolysiscompared to parent against all proteases tested. The BIACORE™ traces forelastase and leucozyme are shown in FIG. 11.

5 μM of each DAB™ was treated with 100 ug/ml sequencing grade trypsinfor 0, 1, 3 and 24 hours. The reaction was inhibited with 25× ROCHECOMPLETE PROTEASE INHIBITOR™ and the reactions were run on a 4-12%NOVEX™ Bis-Tris gel. The gels are shown in FIG. 9.

Example 8

Further Characterization of DOM1h-131-511 and DOM4-130-54 Variants

The DAB™s were first analyzed using Differential Scanning calorimetry(DSC) to determine whether the increase in trypsin resistance correlatedwith an increase in melting temperature (Tm). An increase in trypsinstability does correlate with an increase in Tm (see Table 9)

TABLE 9 Name Tm, ° C. DOM1h-131-511 57.9 DOM1h-131-202 67.5DOM1h-131-203 65.7 DOM1h-131-204 62.3 DOM1h-131-206 64.9 DOM4-130-5454.1 DOM4-130-201 64.7 DOM4-130-202 64.5

The DOM1h-131-511 derived DAB™s were also compared in a MRC-5 cell-basedassay (see Table 10). In this assay, the ability of the DAB™s toneutralize TNFα stimulated IL-8 release was measured to determinewhether the increase in trypsin stability had led to a decrease inefficacy. However, the activity of the trypsin-resistant DAB™s in theassay was substantially unaffected.

TABLE 10 Sample ND50 nM DOM1h-131-511 1.98 DOM1h-131-511 1.71DOM1h-131-511 (230307CE) 1.89 DOM1h-131-203 (230307CE) 2.28DOM1h-131-204 (230307CE) 1.89 DOM1h-131-511 1.46 DOM1h-131-206(230307CE) 0.71

The DOM4-130-54 derived DAB™s were tested in a Receptor Binding Assay tosee if they still had the same ability to inhibit the binding of IL-1 toIL-RI (see Table 11). The activity of the trypsin resistant DAB™s wasunaffected in this assay.

TABLE 11 DAB ™ IC50 (nM) DOM4-130-54 280 pM DOM4-130-201 257 pMDOM4-130-202 254 pM

Example 9

Identification of DOM15-26-555 Variants

DOM15-26-588, DOM15-26-589, DOM15-26-591, and DOM15-26-593 were analyzedin further detail together with their parent and two additional DAB™s,DOM15-26-594 and DOM15-26-595, which were created by mutagenesis tocombine mutations that would have the greatest impact on potency andstability (E6V and F100S/I). Sequences are shown in FIG. 12. Clones werecompared on the BIACORE™ for hVEGF binding at the DAB™ concentration of100 nM after incubation with trypsin at a concentration of 200 μg/ml.The reaction was carried out for three hours and 24 hours at 37° C. withagitation (250 rpm). The BIACORE™ traces of the best clone,DOM15-26-593, and the parent are shown in FIG. 13. Other results arepresented as a chart in FIG. 14. The results clearly show that allvariants are more resistant than the parent to proteolysis after 24hours of trypsin treatment.

Trypsin resistance of DOM15-26-593 and the parent was also examined byrunning treated and un-treated samples on SDS-PAGE. Briefly, proteinswere buffer exchanged to PBS and concentrated to 1 mg/ml. 25 ug ofprotein was mixed with 1 μg of sequencing grade trypsin (Promega) andincubated for 0 hours, 1 hour, 3 hours and 24 hours at 30° C. After thistime, the reaction was blocked with ROCHE COMPLETE PROTEASE INHIBITOR™(1×) and DTT, as well as loading agent, was added. Samples weredenatured for five minutes at 100° C. 15 ug of each sample was loaded onNOVEX™ 10-20% Tricine gels and proteins were stained with SUREBLUE™(1×). The results are shown in FIG. 15. The trypsin resistance profileof DOM15-26-593 in this experiment varied from the profile shown by theBIACORE™ experiment, suggesting that differences in reaction conditionsmay influence the final result of trypsin cleavage. Nonetheless,DOM15-26-593 has better biophysical properties, as well as affinity,than other selected clones, as shown below. A summary of the propertiesof the DOM15-26-555 variants is also shown in Table 12 below.

TABLE 12 Attribute SEC-MALLS Trypsin % DSC BIA- Stability mono- Est. TmRBA CORE ™ % binding DAB ™ mer mw ° C. nM KD nM @ +24 hrs 15-26 0 37-13664 10 28.2 30 15-26-501 0-40 18-290 51 1.14 9.1 5 15-26-555 0 28-78  6311.7 26.1 10 15-26-588 10 33 70 27 59.1 15 15-26-589 90 17 63 1.94 9.665 15-26-591 20 21-234 63 16 38 35 15-26-593 80 17 65 0.323 3.2 8015-26-595 60 17 65 0.828 5 70

Example 10

Identification of DOM15-10 Variants

DOM15-10-11 was analyzed in further detail, together with its parent,DOM15-10. Sequences are shown in FIG. 16. The DAB™s were compared on theBIACORE™ for hVEGF binding at the DAB™ concentration of 100 nM afterincubation with trypsin at a concentration of 200 μg/ml. The reactionwas carried out for 1 hour, 3 hours and 24 hours at 37° C. withagitation (250 rpm). The BIACORE™ traces of these DAB™s are shown inFIG. 17. The result clearly shows that the selected variant is moreresistant than the parent to proteolysis after 24 hours of trypsintreatment.

Trypsin resistance of the lead and the parent was also examined byrunning treated and un-treated samples of SDS-PAGE. Briefly, proteinswere buffer exchanged to PBS and concentrated to 1 mg/ml. 25 μg ofprotein was mixed with 1 μg of sequencing grade trypsin (Promega) andincubated for 0 hours, 1 hour, 3 hours and 24 hours at 30° C. After thistime, the reaction was blocked with ROCHE COMPLETE PROTEASE INHIBITOR™(1×) and DTT, as well as loading agent, was added. Samples weredenatured for five minutes at 100° C. 15 μg of each sample was loaded onNOVEX™ 10-20% TRICENE™ gels and proteins were stained with SUREBLUE™(1×). The results are presented in FIG. 18. In this case, the trypsinresistant profile correlates well with the BIACORE™ trypsin test,showing that the binding activity directly reflects the protein'sintegrity.

Example 11

Further Characterization of DOM15-26-555 and DOM15-10 Variants

The DAB™s were analyzed using Differential Scanning calorimetry (DSC) todetermine whether the increase in trypsin resistance correlated with anincrease in Tm. The results are shown in Table 13. There is acorrelation between the trypsin resistance of DOM15-26-555 variants andmelting temperature. The lead DOM15-26-588 and DOM15-26-593 showedimproved Tm, but the other clones did not. It is worth noting that bothDOM15-26-555 and DOM15-10 parent molecules have much higher Tm at thestart (63.3-63.7° C.) than the DOM4-130-54 and DOM1h-131-511 parentmolecules (Tm at start: 57.9-54.1° C.), but overall the proteaseresistant clones reach a Tm in a similar range (average Tm of 65.1° C.for the DOM1h-131-511/DOM4-130-54 variants and average Tm of 64.9° C.for the DOM15-26-55/DOM15-10 variants).

TABLE 13 Name Tm ° C. DOM15-26-555 63.3 DOM15-26-588 70.1 DOM15-26-58963 DOM15-26-591 63 DOM15-26-593 65 DOM15-10 63.7 DOM15-10-11 63.3

The DAB™s were also compared in a receptor binding assay and BIACORE™kinetics were measured to determine whether the increase in trypsinstability had led to a decrease in efficacy. However, the activity ofthe DAB™s in the assay was substantially unaffected or even improved.The results are presented in Table 14.

TABLE 14 Clone EC₅₀ K_(D) ID (nM) (nM) DOM15- 11.7 26.1 26-555 DOM15- 2759.1 26-588 DOM15- 1.94 9.6 26-589 DOM15- 16 38 26-591 DOM15- 0.323 3.226-593 DOM15- 4.09 15.1 26-594 DOM15- 0.828 5 26-595 DOM15- 10.23 23.610 DOM15- 3.58 14.6 10-11Advantages of an Enhanced Tm

Most proteins—including domain antibodies—exist in two states: a foldedstate (which leads to a biologically active molecule) and an unfoldedstate (which does not bear functional activity). These two statesco-exist at all temperatures and the relative proportion of each stateis usually determined by a constant K that is a function of the kineticconstants of folding and unfolding. The melting temperature is usuallydefined as the temperature at which K=1, i.e. the temperature at whichthe fraction of folded protein is equal to be fraction of unfoldedprotein. The constant K is determined by the stabilizing anddestabilizing intramolecular interactions of a protein and therefore isprimarily determined by the amino acid sequence of the protein.Extrinsic parameters such as temperature, pH, buffer composition,pressure influence K and therefore the melting temperature.

Unfolded proteins are easy targets for degradation mechanisms: (i)exposure of disulfide bonds increase risks of oxidation or reductiondepending on the circumstances, (ii) enhanced backbone flexibilityfavours auto-proteolytic reactions, (iii) exposure of peptide segmentsoffers targets to proteases in vivo, to proteases during productionprocesses and to carry-over proteases during downstream processing andlong-term storage, and (iv) exposure of aggregation-prone segments leadsto inter-molecular aggregation and protein precipitation. In all cases,a loss of protein integrity, protein content and protein activityhappens, thereby compromising efforts to (i) ensure batchreproducibility, (ii) ensure long-term stability on shelf, and (iii) invivo efficacy.

In nature proteins have been designed by evolution to adequately performat body temperature and to be readily replaced via homeostaticmechanisms. Therapeutic proteins manufactured through biotechnologicalprocesses face a different environment: they are frequently produced byrecombinant DNA technology in a foreign host, are expressed at higheramount in large vessels, undergo very important changes in pH or buffercomposition throughout downstream processes and finally are stored athigh concentrations in non-physiological buffers for prolonged period oftime. New delivery techniques (e.g. inhalation, sc patch, slow deliverynanoparticles) are also adding on the stress undergone by therapeuticproteins. Finally the advent of protein engineering techniques hasresulted in the production of enhanced or totally novel therapeuticproteins. Because most engineering techniques are in-vitro basedtechniques aimed at altering or creating new amino acid sequences,evolution processes that have gradually improved biological proteins donot take place, hence resulting in proteins of sub-optimal performanceswith regards to stress resistance.

The technique of the present invention aims at reproducing one of theconditions faced by proteins throughout Darwinian evolution. Peptides orpolypeptides, e.g. immunoglobulin single variable domains are infusedwith proteases that play a major role in tissue remodelling and proteinhomeostasis. Any particular mutation that may result in a protein withan improved fit to its function is also tested for its ability to fitwithin the environment it is performing in. This process is reproducedin one embodiment of the present invention: a repertoire of peptide orpolypeptide variants is created and exposed to a protease. In a secondstep, the repertoire of variants is contacted with a specific target.Only those protein variants that have sustained degradation by theprotease are able to engage with the target and therefore recovered,e.g., by a simple affinity purification process named ‘biopanning’. Thesystem offers a number of advantages in comparison to in vivo processes:the protein repertoire can be faced with a wider range of conditions,e.g. a range of proteases, at higher concentrations, for longer times,in different buffers or pHs and at different temperatures. Thus this invitro technology offers a means to design proteins that may perform andremain stable in a wider range of environments than those they originatefrom. Clearly this offers multiple advantages for the biotechnologicalindustry and for the area of therapeutic proteins in particular.

Example 12 PK Correlation Data for Protease Resistant Leads

The parent DAB™ and a protease-resistant DAB™ in each of the four DAB™lineages, were further evaluated in vivo (see Table 15 below for listand details)

TABLE 15 Resistance Tm Activity ID as Fc Lineage DAB ™ ID to trypsin (°C.) (nM) fusion DOM4-130 DOM4-130- Good 54 0.128* DMS1541 54 DOM4-130-Very high 64 0.160* DMS1542 202 DOM1h- DOM1h-131- Good 57 0.048† DMS1543131 511 DOM1h-131- Very high 64 0.047† DMS1544 206 DOM15-10 DOM15-10 Low64 0.913† DMS1546 DOM15-10- High 63 0.577† DMS1531 11 DOM15-26 DOM15-26-Low 52 0.330† DMS1545 501(*) DOM15-26- High 65 0.033† DMS1529 593 (*)asdetermined by MRC5/IL-a bioassay; †as determined by RBA assay

Note: DOM15-26-501 is a parent version of DOM15-26-555 exemplified abovein this patent application. DOM15-26-555 has one germline amino acidmutation in CDR1 (134M). DOM15-26-501 has a lower melting temperaturethan DOM15-26-555 (52C v 63.3C) and an increased susceptibility todigestion by trypsin. DOM15-26-501 was chosen over DOM15-26-555 for thePK study as it is a better representative for poor stability incomparison to DOM15-26-593.

We can translate the resistance as follows:

1 is low

2 is moderate

3 is good

4 is high

5 is very high

Then this means that the trypsin resistance of the parent molecules is:

DOM4-130-54 is Good

DOM1h-131-511 is Good

DOM15-10 is Low

DOM15-26-501 is Low

As for the selected leads:

DOM4-130-202 is Very high

DOM1h-131-206 is Very high

DOM15-10-11 is High

DOM15-26-593 is High

Because domain antibodies are small in size (12-15 kDa) they are rapidlycleared from the circulation upon iv or sc injection. Indeed the renalglomerular filtration cut-off is above 50 kDa and therefore smallproteins such as DAB™s are not retained in the circulation as they passthrough the kidneys. Therefore, in order to evaluate the long termeffects of resistance to proteases in vivo, we tag domain antibodieswith a moiety that increases systemic residence. Several approaches(e.g. PEG, Fc fusions, albumin fusion, etc) aiming at extendinghalf-life have been reported in the literature. In this application thedomain antibodies have been tagged (or formatted) with the Fc portion ofthe human IgG1 antibody. This format offers two advantages: (i) themolecular size of the resulting dAb-Fc is ˜75 kDa which is large enoughto ensure retention in circulation, (ii) the antibody Fc moiety binds tothe FcRn receptor (also know as “Brambell” receptor). This receptor islocalized in epithelial cells, endothelial cells and hepatocytes and isinvolved in prolonging the life-span of antibodies and albumin: indeedupon pinocytosis of antibodies and other serum proteins, the proteinsare directed to the acidified endosome where the FcRn receptorintercepts antibodies (through binding to the Fc portion) before transitto the endosome and return these to the circulation. Thus by tagging theFc portion to the DAB™, it is ensured that the DAB™s will be exposed forlong period to two at least compartments—the serum and the pre-endosomalcompartments, each of which containing a specific set of proteolyticenzymes. In addition, the FcRn receptor mediates transcytosis wherebyFc-bearing proteins migrate to and from the extravascular space.

Formatting with Fc was accomplished by fusing the gene encoding the VHand VK DAB™s to the gene encoding the human IgG1 Fc, through a shortintervening peptide linker (in bold):

For a VH DAB™ (underlined):

EVQ . . . . . . GQGTLVTVSSASTHTCPPCPAPELLGGP (SEQ ID NO: 260) . . . (hIgGlFc) . . . PGK*

For a VK DAB™ (underlined):

DIQ . . . . . . . . . GQGTKVEIKRTVAAPSTHTCPPCPAPELLGGP (SEQ ID NO: 261) . . . (hIgGlFc) . . . PGK*

Material was produced by transient transfection of HEK293/6E cells using293-FECTIN™ (Invitrogen) according to standard protocols. These cellsare designed for high-level transient expression when used inconjunction with the pTT series of vectors (Durocher et at 2002). Thusthe DAB™ genes were cloned into a modified pTT5 vector (pDOM38) togenerate the Fc fusion expression vector (see FIG. 21). The supernatantfrom the transfected cells was harvested at 5 days post-transfection,clarified by centrifugation and filtered through a 0.2 μm filter. ThedAb-Fc fusion proteins were purified by capture onto Protein-ASTREAMLINE™ resin (GE Healthcare). Protein was eluted from the column in10 mM sodium citrate pH3, followed by the addition of and 1M sodiumcitrate pH6, to achieve a final composition of 100 mM sodium citrate pH6.

The dAb-Fc molecules were tested for in vivo half life in the rat at atarget dose of 5 mg/kg into female Sprague-Dawley rats (n=3 per group).It should be noted that the target dose vastly exceeds targetconcentration in rats, so it is expected that differences in affinitiesbetween parent DAB™s and trypsin-resistant DAB™s (see example 11) willnot impact on the fate of the molecules in vivo. Hence differences in PKprofiles between DAB™s are expected to reflect on an antigen-independentelimination process.

Blood samples were taken after 0.03, 1, 4, 8, 24, 48, 72, 96, 120 and168 hours post administration. After clot formation, serum was withdrawnand then tested in hIL-1R1, TNFR1 or VEGF antigen capture assays:

hIL-1R1 Antigen Capture Assays:

Coat with 4 ug/mL anti-hIL-1R1

Block

Add 500 ng/mL shIL-1R1

Add samples

Detect with anti-human Fc HRP @1:10,000

TNFR1 Antigen Capture Assays:

Coat with 0.1 ug/mL sTNFR1

Block

Add samples

Detect with anti-human Fc HRP @1:10,000

VEGF Antigen Capture Assays:

Coat with 0.25 ug/mL VEGF

Block

Add samples

Detect with anti-human Fc HRP @1:10,000

Raw data from the assays were converted into concentrations of drug ineach serum sample. The mean μg/mL values at each time point were thenanalysed in WINNONLIN™ using non-compartmental analysis (NCA). The PKprofiles of each dAb-Fc pair are shown in Table 16 which summarises thedetermined PK parameters.

TABLE 16 AUC/D (0-inf) Half Life (hr * μg/mL)/ % AUC ID DAB ™ (hr)(mg/kg) Extrapolated DMS1541 4-130-54 93.2 691.5 22.7 DMS1542 4-130-202176.8 710.1 49 DMS1543 1h-131-511 140.8 1807.5 40 DMS1544 1h-131-206158.6 2173.0 43.6 DMS1546 15-10 43.2 324.6 3.8 DMS1531 15-10-11 56.6770.5 n.d. DMS1545 15-26-501 12.9 89 5.1 DMS1529 15-26-593 86.2 804.721.0

The results clearly indicate that—whilst the PK profiles of the dAb-Fcpairs 4-130-54 to 1h-131-206 are almost superimposable—the profiles varywidely with the other pairs. The effects are mostly visible when AUC/Dis considered: the AUC/D of 15-10 is only 42% of that of 15-10-11. TheAUC/D of 15-26-501 is only 11% of that of 15-26-593. These importantdifferences also impact (to a lesser extent) half-lives: 43.2 h versus56.6 h for 15-10 and 15-10-11, respectively. A greater difference isseen with the DOM15-26 lineage: 12.9 h versus 86.2 h for 15-26-501 and15-26-593, respectively. Indeed for a good PK analysis usingnon-compartmental analysis, there should be at least 4 data points usedto fit the linear regression slope and the period of time over which thehalf life is estimated should be at least 3 times that of the calculatedhalf life.

In light of the biophysical properties described in the examples herein,it appears that the ability of any given DAB™ to resist degradation bytrypsin is correlated with the ability of the dAb-Fc fusion to circulatefor a longer period in the rat serum. Indeed as shown in the examples,such as Example 10, DOM15-10 and DOM15-26-501 are the most degradableDAB™s: incubation of 25 ug DAB™ in the presence of 1 ug of trypsin at30° C. for ˜3 h resulted in complete degradation. All other DAB™s inthis study (whether they had been selected with trypsin (i.e.DOM15-10-11, DOM15-26-593, DOM4-130-202 and DOM1h-131-206) or whetherthey already had some trypsin resistance as parent molecules(DOM4-130-54 and DOM1h-131-511)) have comparable PK profile in rats whenre-formatted into dAb-Fc molecules. Thus, the present PK study suggeststhat susceptibility to proteolysis has its biggest impact on the in vivostability of DAB™s when those DAB™s have very low resistance toproteolysis. It also shows that—beyond a certain level—furtherincrements in resistance to degradation by trypsin (e.g. DOM4-130-206 vDOM4-130-54) do not significantly add up to the ability of the dAb-Fcmolecule to further slow down elimination in vivo.

In three cases, selection in the presence of trypsin resulted in newmolecules with increased thermal stability (defined by the meltingtemperature): DOM4-130-202, DOM1h-131-206 and DOM15-26-593. The PK studyindicates that—in the present dataset—melting temperature is not anadequate parameter to rationalize the observed PK profiles: indeedDOM15-10 has a higher Tm than DOM15-10-11 and yet is more rapidlycleared than DOM15-10-11 from the circulation. Elsewhere, the two DAB™sof the DOM4-130 lineage have markedly different Tm (by 10° C.) and yetshow almost identical stability in vivo when formatted into dAb-Fcmolecules. It should be noted that melting temperature is not per seexcluded as key parameter to predict in vivo stability. It just happensthat with the present dataset, large Tm differences (from 54° C. andabove) have not a significant impact on the fate of DAB™s in vivo. Thisdoesn't exclude the possibility that at melting temperature lower than54° C., the in vivo stability of DAB™s may correlate with thermalstability, or perhaps even with thermal stability and resistance toproteases altogether.

Example 13

Trypsin Selections on DOM10-53-474

Trypsin Stability of Purified DOM10-53-474:

DOM10-53-474 is a domain antibody which binds to IL-13 with a highpotency. To assess the stability of this DAB™ in the presence oftrypsin, purified DAB™ was digested with trypsin for increased timepoints and run on a gel to examine any possible protein degradation. 25μl of purified DOM10-53-474 at 1 mg/ml was incubated with 1 μl ofsequencing grade trypsin at 1 mg/ml at 30° C., resulting in molecularratio of 25:1 DAB™:trypsin. DAB™ was incubated with trypsin for 1 h, 4 hand 24 h and the protease activity was neutralised by addition of 4 μlof ROCHE COMPLETE PROTEASE INHIBITORS™ followed by incubation on ice.Time 0 sample was made by adding protease inhibitors to DAB™ withoutadding trypsin. 2 μl of sample was subsequently analysed byelectrophoresis using LABCHIP™ according to manufacturers instructions.

FIG. 22 shows a gel run with DOM10-53-474 incubated with typsin forincreased time points. For comparison one of the trypsin stable DAB™s,DOM15-26-593 was also treated with trypsin as explained above and wasrun alongside. As shown in the figure, DOM15-26-593 looks stable evenafter 24 h incubation with trypsin. However, DOM10-53-474 is degraded toa certain extent after 24 h, but looking stable at 1 h and 4 h timepoints. These data suggests that DOM10-53-474 is resistant todegradation by trypsin to a certain extent, but is not as stable as oneof the most trypsin stable DAB™s DOM15-26-593.

Trypsin Stability of Phage-Displayed DOM10-53-474:

To assess the trypsin stability of phage displayed DOM10-53-474, thegene encoding DOM10-53-474 was cloned into Sal/Not sites of pDOM33 (FIG.50) and phage produced according to standard techniques. Phage waspurified by PEG precipitation, resuspended in PBS and titered.

Phage displayed DAB™s were incubated with trypsin for different timepoints to evaluate trypsin resistance. Following incubation withtrypsin, stability was measured by titre analysis following infection ofexponentially growing E. coli TG1 cells.

100 μl of phage was incubated in 100 μg/ml trypsin for 1 h, 2 h, 4 h andovernight at 37 C, in a shaking incubator. Trypsin activity was blockedwith ROCHE COMPLETE PROTEASE INHIBITOR™ (×2) and then phage was dilutedin 2% marvel in PBS, incubated with 10 nM biotinylated IL-13 for onehour at room temperature. Streptavidin-coated beads (DYNABEADS™ M-280(Invitrogen) that were pre-blocked for one hour at room temperature with2% MARVEL™ in PBS was added, and the mixture was then incubated for 5minutes at room temperature. All of the incubation steps with DYNABEADS™were carried out on a rotating wheel. Unbound phage was washed away bywashing the beads eight times with 1 ml of 0.1% TWEEN-20™ in PBS. Boundphage was eluted with 0.5 ml of 0.1M Glycine pH 2.2 and neutralized with100 μl of 1M Tris-HCL pH 8.0. Eluted phage was used to infectexponentially growing TG1 (1 h at 37° C.) and plated on tetracyclineplates. Plates were incubated at 37° C. overnight and colony counts weremade. Phage output titres following digestion with trypsin is summarisedin Table 17. Phage titres decreased when incubated with trypsin forincreased time points. After 24 h incubation all phage was digested.

TABLE 17 Output titres of trypsin selections performed on phagedisplayed DOM-10-53-474 parent: Length of trypsin incubation Trypsinconcentration Titre No trypsin control — 3 × 10⁷ 1 h 100 μg/ml 1 × 10⁷ 2h 100 μg/ml 7 × 10⁶ 4 h 100 μg/ml 5 × 10⁶ overnight 100 μg/ml 0Selection of DAB™s More Resistant to Trypsin:

In order to select for DAB™s which are more resistant to degradation bytrypsin, random mutations were introduced to gene encoding DOM10-53-474by PCR using STRATAGENE MUTAZYME II™ kit, biotinylated primers and 5-50pg of template for 50 μl reaction. After digestion with SalI and NotI,inserts were purified from undigested products with streptavidin coatedbeads and ligated into pDOM33 at the corresponding sites. E. Coli TB 1cells were transformed with purified ligation mix resulting in an errorprone library of DOM10-53-474. The size of the library was 1.9×10⁹ andthe rate of amino acid mutation was 1.3.

Three rounds of selections were performed with this library to selectfor DAB™s with improved protease resistance. First round of selectionwas performed only with antigen without trypsin treatment to clean upthe library to remove any clones that no longer bound antigen with highaffinity. Selection was carried out at 10 nM IL-13. The outputs fromround one were 2×10⁹ compared to input phage of 6×10¹⁰ indicating thatmajority of library bound antigen with high affinity.

The second and third rounds of selections were performed with 1 nMbiotinylated IL-13. Prior to panning on IL-13, phage was incubated with100 μg/ml of trypsin at 37° C. in a shaker (250 rpm). For second roundselection, trypsin incubation was carried out for 1 h either at roomtemperature or at 37° C. The outputs from round 2 selection is shown inTable 18:

TABLE 18 Output phage titres following second round selection. Trypsintreatment Titre No treatment 1 × 10⁸ 1 h room temperature 5 × 10⁷ 1 h37° C. 2 × 10⁷

Phage outputs from round 2 selection with 1 h trypsin treatment at 37°C. was used as the input for 3^(rd) round selection. For 3^(rd) roundselection, phage was treated with 100 μg/ml trypsin but for longer timepoints: 2 h at 37° C., 4 h at 37° C., overnight at room temperature orovernight at 37° C. The output titres for 3^(rd) round selection aresummarised in Table 19:

TABLE 19 Output phage titres following third round selection Trypsintreatment Titre No trypsin 1.3 × 10⁸ 2 h at 37° C. 1.9 × 10⁷ 4 h at 37°C.   2 × 10⁶ Overnight at room temperature   4 × 10⁷ Overnight at 37° C.2.1 × 10⁶

Several clones from each selection outputs from round 1, 2 and 3 weresequenced to assess the sequence diversity. Following first round ofselection without trypsin treatment, 50% of the selection outputs hadparent DOM10-53-474 sequence. After 2^(nd) round of selection,percentage of parent increased to 75%. After 3^(rd) round of selection,percentage of parent increased to 80%.

This data indicate that DOM10-53-474 is already resistant to degradationby trypsin and not many new clones can be selected from these trypsinselections. FIG. 22 showed that when purified protein was digested withtrypsin, DOM10-53-474 was not completely digested even after overnighttrypsin treatment. However to see whether there are any new clones thatare more trypsin resistant than DOM10-53-474 in selection outputs,selection 3 output where phage was treated overnight with trypsin at 37°C. was sub-cloned into pDOM5. Hundred clones were then sequenced to lookfor any trypsin resistant clones. Out of hundred clones analysed, only26 clones had new sequences, however none of these clones had mutationsat trypsin cleavage sites (Lysine or Arginine) suggesting that theseclones are not more resistant to trypsin than DOM10-53-474.

Example 14

Storage and Biophysical Improvements Introduced into the Lead DOM0101(Anti-TNFR1) DAB™s by Phage Selections in the Presence of Trypsin

To improve the protease resistance of the lead molecule DOM1h-131-511,phage selections in the presence of trypsin were carried out asdescribed earlier. The method produced a range of clones with improvedtrypsin stability compared to the parental DOM1h-131-511 molecule. Twoclones, DOM1h-131-202 and DOM1h-131-206 were selected for furthercharacterisation as they showed the most significant improvement to theaction of trypsin. Further work as outlined below shows that with theimproved resistance to the action of trypsin there are other beneficialeffects, primarily on the stability of the molecules to shear andthermal stress. These two parameters are central to increasing thestorage and shelf life stability of biopharmaceutical products.

Production of Lead DOM0101 DAB™s in Pichia pastoris:

The genes encoding the primary amino acid sequence of the three leadmolecules was used to produce secreted protein in Pichia pastoris. Thethree synthetic genes (DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206)were cloned into the expression vector pPIC-Zα and then transformed intotwo Pichia strain, X33 and KM71H. The transformed cells were plated outonto increasing concentrations of ZEOCIN™ (100, 300, 600 and 900 μg/ml)to select for clones with multiple integrants. Several clones were thenscreened in 2 L flasks to identify high expressing cell lines. The bestexpressing clones were then used to produce material at 5 L scale infermenters.

Protein Purification and Material Characterization:

In order to produce high quality material for characterisation andfurther stability studies, a downstream purification process was devisedusing a mixed modal charge induction resin (CAPTO™ MMC) as the primarycapture step followed by anion exchange (Q Sepharose). Withoutsignificant optimisation, this allowed the recovery of ˜70% of theexpressed DAB™ at a purity of ˜95%. The material was characterised foridentity using electrospray mass spectrometry, amino terminal sequencingand isoelectric focusing and for purity using SDS-PAGE and SEC (sizeexclusion chromatography).

Protein Identity:

The amino terminal sequence analysis of the first five residues of eachprotein, was as expected (EVQLL . . . ) (SEQ ID NO: 259). Massspectrometry was performed on samples of the proteins which had beenbuffer exchanged into 50:50 H₂O:acetonitrile containing 0.1% glacialacetic acid using C4 ZIP-TIPS™ (Millipore). The measured mass for eachof the three proteins was within 0.5 Da of the theoretical mass based onthe primary amino acid sequence (calculated using average masses) whenallowing for a mass difference of −2 from the formation of the internaldisulphide bond. IEF was used to identify the proteins based on their pIwhich was different for each protein.

Protein Purity:

The three proteins were loaded onto non-reducing SDS-PAGE gels in 1 μgand 10 μg amounts in duplicate. A single band was observed in allinstance.

Size exclusion chromatography was also performed to demonstrate levelsof purity. For size exclusion chromatography (SEC) 100 μg of eachprotein were loaded onto a TOSOH G2000 SWXL™ column flowing at 0.5ml/min. Mobile phase was PBS/10% ethanol. The percentage of monomer wasmeasured based on the area under the curve (see FIG. 23).

Comparison of Stability of DOM1h-131-511, -202 and -206Assessment ofProtease Stability:

The protease stability of DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206was assessed by BIACORE™ analysis of the residual binding activity afterpre-incubation for defined timepoints in excess of proteases.Approximately 1400 RU of biotinylated TNFR1 was coated to a streptavidin(SA) chip. 250 nM of DOM1h-131-511, DOM1h-131-202 and DOM1h-131-206 wasincubated with PBS only or with 100 ug/ml of trypsin, elastase orleucozyme for 1, 3, and 24 hour at 30° C. The reaction was stopped bythe addition of a cocktail of protease inhibitors. The DAB™/proteasemixtures were then passed over the TNFR1 coated chip using referencecell subtraction. The chip surface was regenerated with 10 ul 0.1Mglycine pH 2.2 between each injection cycle. The fraction ofDOM1h-131-511, DOM1h-131-202 and DOM1h-131-206 bound to human TNFR1 (at10 secs) pre-incubated with proteases was determined relative to DAB™binding without proteases. BIACORE™ runs were carried out at 25° C. Thedata below was produced from three independent experiments. The bargraph indicates mean values and the error bars indicate standarddeviation of the results (FIG. 24).

It was found that DOM1h-131-202 and DOM1h-131-206 were shown to havegreater resistance to proteolytic degradation by trypsin, elastase orleucozyme in comparison to DOM1h-131-511. The difference betweenDOM1h-131-202 and DOM1h-131-206 in comparison to DOM1h-131-511 is mostpronounced after 1 hr with trypsin and after 3 hrs with elastase orleucozyme. There is a trend that DOM1h-131-206 is slightly more stablecompared to DOM1h-131-202 in most of the conditions tested.

Thermal Stability of the DAB™s as Determined Using DSC:

In order to determine at which pH the lead molecules had the greateststability, differential scanning calorimeter (DSC) was used to measurethe melting temperatures (T_(m)) of each DAB™ in Britton-Robinsonbuffer. As Britton-Robinson is made up of three component buffer systems(40 mM of each of acetic, phosphoric and boric acid), it is possible toproduce a pH range from 3-10 in the same solution. The theoretical pIwas determined from the proteins primary amino acid sequence. From theDSC, the pH at which the DAB™s had their greatest intrinsic thermalstability was found to be pH 7 for DOM1h-131-202, pH 7-7.5 forDOM1h-131-206 and pH 7.5 for DOM1h-131-511. For all subsequent stressand stability work the following pHs were used for each DAB™; forDOM1h-131-202 and GSK1995057A DOM1h-131-206 pH 7.0 and for DOM1h-131-511pH 7.5 in Britton-Robinson buffer. The results are summarised in Table20.

TABLE 20 Summary of the pH and T_(m)s of DOM1h-131-202, DOM1h-131-206and DOM1h-131-511 as determined by DSC in Britton-Robinson buffer at 1mg/ml. The temperature was ramped at 180° C./hour. pH that givesgreatest Tm (° C.) of the DAB ™ at DAB ™ intrinsic thermal stability thegiven pH DOM1h-131-202 7.0 68.6 DOM1h-131-206 7.0-7.5 65.8 DOM1h-131-5117.5 58.0

Two Week Thermal Stability Testing

The ability of a protein to endure prolonged periods of time at elevatedtemperatures is usually a good indication of its stability. Under theseconditions, protein may undergo several physical processes such asaggregation or chemical modification. The DAB™s (at 1 mg/ml) wereincubated at 37 and 50° C. for 14 days in Britton-Robinson buffer. SECwas used to determine how much monomer was left in solution over the 14day period (FIG. 25).

From FIG. 25 it can be seen that both DOM1h-131-202 and DOM1h-131-206are significantly more stable than DOM1h-131-511 to thermal stress.Exposing proteins to elevated temperatures, such as 37 and 50° C., areroutinely used to give an indication on a drug's long term shelf-life.These higher temperatures are used to accelerate the normal processassociated with long term storage at room temperature such asdeamidation, oxidation or aggregation. The level of aggregationformation in solution can also be monitored using SEC (FIG. 26A to I).After 14 days at 37° C., the loss of DOM1h-131-511 from solution can beattributed to both precipitation and the formation of higher orderedaggregates as determined by SEC (FIG. 26B). A significantly lower lossin protein is also seen with both DOM1h-131-202 and DOM1h-131-206 at 37°C. after 14 days with very little or no substantial increase inaggregate formation, especially in the case of DOM1h-131-206 (FIG. 26H).At 50° C., the difference between the molecules is even more pronounced,with DOM1h-131-206 showing better stability at the higher temperaturethan DOM1h-131-202 after 14 days, showing significantly reducedformation of higher molecular weight aggregates (FIG. 26). Relative tothe t=0, DOM1h-131-206 shows only a small increased in aggregateformation after 14 days (FIG. 26I), whereas DOM1h-131-511 has all butprecipitated out of solution (FIG. 26C).

This shows that the changes introduced into the DAB™ by the trypsinselections, e.g. the improved thermal stability, has significantlyimproved the protein storage stability at 37 and 50° C. BothDOM1h-131-202 and more significantly DOM1h-131-206, clearly haveimproved solution stability and lower tendency to form aggregates atelevated temperatures which can directly be translated to improved longterm storage stability at more relevant temperatures such +4° C. androom temperature.

Samples from 24 hr, 48 hr, 7 days and 14 days time points from thethermal stress experiment were then analysed by IEF to see if theproteins had undergone any biophysical changes which would affect theoverall charge of the protein (FIG. 27).

Again both DOM1h-131-202 and DOM1h-131-206 show no significant changesat 37° C. compared to DOM1h-131-511. With DOM1h-131-511a faint secondband appears at 37° C. after 24 hrs. It is believed this extra bandingis due to dimerisation of the protein, thus masking charge and producingtwo populations of molecules. At 50° C. the difference between themolecules is more pronounced, DOM1h-131-206 clearly shows no significantchanges at the elevated temperature whereas DOM1h-131-202 is showingsome sign of modification after 24 hr. The majority of DOM1h-131-511 islost by precipitation after 48 hr in Britton-Robinson.

The T=0, 7 and 14 day time points at 50° C. were analysed by the TNFR-1RBA to determine the functionality of the protein after exposure to hightemperatures (FIG. 28). The assay is currently not as sensitive as SECor IEF at detecting subtle changes to the molecule due to stress, but itcan be used show that the DAB™ can still bind to the antigen.

The shift in the curve to the left for DOM1h-131-511 reflects the factthat the majority of the DAB™ has been lost due to precipitation. Thematerial that is left in solution is still able to bind antigen. Asshown in FIG. 25, the majority of both DOM1h-131-202 and DOM1h-131-206are able to be maintained in solution even after 14 days. The RBA showsthat all the soluble protein is still functional and able to bind toTNFR1.

Storage Stability Testing at High Protein Concentrations:

Experiments were carried out to investigate the storage stability at +4°C. at very high protein concentrations to see how each moleculeperformed under these conditions. All the lead DAB™s were concentratedin centrifugal VIVASPIN™ concentrators (5K cut-off) in Britton-Robinsonbuffer at their most stable pH, to ˜100 mg/ml. The samples at ˜100 mg/mlwere then left at +4° C. for 7 days and then analysed by SEC to see ifany other physical changes had occurred to the samples during storage athigh concentrations (FIG. 29). The samples were diluted to ˜1 mg/mlbefore being run on the SEC column in 1×PBS 10% ethanol (v/v).

From the SEC traces it can be seen that neither DOM1h-131-202 norDOM1h-131-206 show any significant increase in the formation ofaggregates after 7 days, where as there is ˜2% reduction in the monomerconcentration for DOM1h-131-511.

Nebuliser Delivery of the Lead DAB™s:

For early stage toxicology and clinical work, the DAB™s will beformulated as a liquid and delivered via a nebulising device. Dependingon the device (e.g., ultrasonic, jet or vibrating mesh), the DAB™ willexperience a degree of shear and thermal stress as it was nebulised toform a aerosol of defined particle size. As both DOM1h-131-202 and -206have higher T_(m)'s and showed considerably improved stability tothermal stress compared to DOM1h-131-511, all the DAB™s were tested intwo nebuliser devices to see how they responded to shear/thermal stressinduced during nebulisation. Both the protein from the nebulised aerosoland the remaining DAB™ in the device (i.e. in the cup) were thenanalysed by SEC to determine the amount of aggregation generated duringthe process.

All the molecules were tested in Britton-Robinson buffer at their moststable pH. The DAB™s were tested in both the EFLOW RAPID™ (vibratingmesh) and PARI LC+™ (jet nebuliser) with run time of 3.5 minutes at aprotein concentration of 5 mg/ml and the particle size distributiondetermined using a Malvern Spraytec. The results are shown in FIG. 30.For good delivery and distribution into the deep lung, the idealparticle size is <5 μm. All the DAB™s give comparable levels of particlesizes that were less than 5 μm in Britton-Robinson buffer. Theconcentration of the DAB™ in the cup of the device was determined byA₂₈₀ measurements before and after nebulisation (data not shown). It wasfound that the protein concentration did not change significantlyindicating that neither the protein nor vehicle are preferentiallynebulised during delivery.

Samples of the DAB™s nebulised in Britton-Robinson buffer were run onSEC to determine if during delivery the protein had undergone anyphysical changes. FIG. 31 shows the relative percentage change in eitherthe cup or the aerosol as determined by SEC. It can be seen that bothDOM1h-131-202 and DOM1h-131-206 undergo relative small changes in theconcentration of monomer relative to DOM1h-131-511. This demonstratesthat both DOM1h-131-202 and DOM1h-131-206 with their improved T_(m)'shave less propensity to aggregate during nebulisation.

FIG. 32 shows the actual SEC traces for DOM1h-131-206 and DOM1h-131-511in Britton-Robinson buffer post nebulisation and demonstrates that therelative loss in monomer (FIG. 31) is due to dimer formation. This againprovides further supporting evidence to the theory that the greaterthermal stability shown by DOM1h-131-202 and DOM1h-131-206 can preventsignificant aggregation even in an unoptimised formulation buffer.

For toxicology and safety assessment work, it is necessary to deliverythe DAB™ at significantly higher levels into the animal than thetherapeutic doses given to patients. This can only be achieved by usingsignificantly higher protein concentrations and/or delivering the DAB™over a prolonged period of time. As it had already been shown thatDOM1h-131-511 forms aggregates on nebulisation at 5 mg/ml over 3.5 mins,DOM1h-131-206 was tested at 40 mg/ml in PBS and nebulised using the PARILC+™ for up to 1 hour. Samples from the cup and aerosol were taken atthe time points to throughout the run to see if the prolong nebulisationcaused the DAB™s to aggregate due to shear or thermal stress asdetermined by SEC and the protein concentration (A280 nm measurements).Table 21 shows the protein concentration of the DAB™ both in the cup andaerosol as determined by A280.

TABLE 21 Measured protein concentration of DOM1h-131-206 as determinedby A280 absorbance readings for both the cup and aerosol duringnebulisation of the DAB ™ at ~40 mg/ml using the PARI LC+ ™. Allowingfor dilution errors and instrumental error the sample concentration doesnot change after nebulising the DAB ™ over 1 hr. Time Cup Sample (Mins)(mg/ml) Aerosol Sample (mg/ml) 1 43.8 43.4 29 44.5 43.5 59 44.6 44.1

From Table 21 it can be seen that the concentration of the protein didnot significantly change during the run, demonstrating that there was nosignificant loss of the protein due to aggregation. FIG. 33 shows thatover the period of 1 hour of nebulisation, DOM1h-131-206 does not formany higher ordered aggregates such as dimers as determined by SEC. Thisclearly demonstrates that the improved biophysical properties, asintroduced into the molecule by trypsin selections, significantlyincreases the DAB™'s resistance to shear and thermal stress and thatthis can be directly correlated to improved storage shelf-life and theability to nebulise the protein so that higher ordered aggregates do notform.

Solution State of the Lead DAB™s:

Since the major route of degradation for all the three lead DAB™sappears to be self-association leading initially to dimerisationfollowed by further aggregation and ultimately precipitation, the threelead molecules were investigated by Analytical Ultra-Centrifugation(AUC) to determine the degree of self-association. The proteins wereinvestigated by two methods, sedimentation equilibrium and sedimentationvelocity.

For the sedimentation equilibrium method the three samples were run atthree different concentrations ranging from 0.5 mg/ml to 5 mg/ml withcentrifugation effects using three different rotor speeds. By thismethod it was determined that DOM1h-131-511 is a stable dimer (26.1-34.4kDa), DOM1h-131-202 is monomer/dimer equilibrium (22.7-27.8 kDa) with arelatively stable dimeric state at the concentrations measured withK_(d)=1.3 μM and DOM1h-131-206 is predominantly monomeric (15.4-17.9kDa) with a K_(d) for the monomer to dimer association of 360 μM.

By the sedimentation velocity method all samples showed some degree ofdissociation upon dilution. From the results obtained, shown in FIG. 34,the sedimentation coefficient observed for DOM1h-131-511 is indicativeof higher order aggregates and the peak shift upon dilution is anindication of dissociation of these aggregates. The protein aggregationand dissociation cancel each other out which can give the impression ofbeing a stable dimer as observed by sedimentation equilibrium. Thesedimentation coefficients observed for DOM1h-131-202 are indicative ofa rapid dynamic equilibrium and therefore the monomer and dimer peakscould not be separated from each other, giving the single peak with ahigher sedimentation coefficient than is appropriate for the mass of thesample. This result agrees with the result obtained by the sedimentationequilibrium method and the dissociation constant was measured as being 1μM. DOM1h-131-206 was determined to be more monomeric than the other twosamples, having a sedimentation coefficient of 1.9 s as compared to 2.5s for the other two samples. This data agrees well with thesedimentation equilibrium data. At the concentrations measured, ˜10-foldbelow the K_(d) of 360 μM, the sample is predominantly monomeric.

Example 15

Potency Enhancement of the DOM15-26-593 DAB™

An example of the enhancement of potency in VEGFR2Receptor Binding Assayof the DOM15-26-593 DAB™ over DOM15-26 parent is shown in FIG. 40. Inthis assay, the ability of a potential inhibitor to prevent binding ofVEGF to VEGFR2 is measured in a plate-based assay. In this assay aVEGFR2-Fc chimera is coated on a 96-well ELISA plate, and to this isadded a predetermined amount of VEGF that has been pre-incubated with adilution series of the test DAB™. Following the washing-off of unboundprotein, the amount of VEGF bound to the receptor is detected with ananti-VEGF antibody, the level of which is determined colorimetrically. Adose-response effect is plotted as percentage inhibition of VEGF bindingas a function of test substance concentration. An effective inhibitor istherefore one that demonstrates substantial blocking of ligand bindingat low concentrations.

FC Fusions Potency and Half Life:

The therapeutic potential of VEGF blockade in the treatment of tumourshas been realised for over 30 years. The chronic nature of cancerdictates that biopharmaceuticals require a long serum half life tomediate their effects, and this is not consistent with the rapidclearance of free DAB™s from the circulation by renal filtration. Toassess the utility of the VEGF DAB™s as anti-angiogenics for thetreatment of cancer, the lead domain antibodies were formatted asfusions with wild type human IgG1 Fc via a hybrid linker so as to form abivalent molecule with a serum half life extended by the use ofFcRn-mediated antibody salvage pathways.

In this Fc fusion format, the potency of the lead trypsin selected DAB™,DOM15-26-593 was compared with the initial parent DAB™ (DOM15-26) & thetrypsin labile DAB™ (DOM15-26-501) using the assay described previously.The results are shown in the Table 22 below:

TABLE 22 Potency (RBA) & half life characteristics of DOM15-26 leads inthe Fc fusion format DAB ™ Fc Potency (nM) T½b (hrs) DOM15-26 hIgG10.506 ND DOM15-26-501 hIgG1 0.323 12.9 DOM15-26-593 hIgG1 0.033 84.6

It can be seen from these results that in the dimeric Fc fusion format,affinity & potency are enhanced in relation to the free DAB™s due to theeffect of avidity. It is clear that the potency enhancement obtained inDOM15-26-593 by virtue of trypsin selection is maintained and is evenmore pronounced in this Fc format. Furthermore, the improvements inthermal and protease stability translate into profound changes in the invivo pharmacokinetic behaviour of the molecules. The improvement in theelimination half life (see FIG. 41) of DOM15-26-593 compared withDOM15-26-501 is likely to be a direct consequence of the increasedstability of the DAB™, rendering it more resistant to the degradativeprocesses that occur within the endosomal compartment. It is also to beexpected, therefore, that DAB™s with enhanced protease stability areable to persist for longer in other biological compartments such as theserum, mucosal surfaces and various tissue compartments whereproteolysis is an active process involved in the turnover of biologicalmolecules.

Pharmacokinetic Clearance Profiles:

Pharmacokinetic clearance profiles of DOM15-26-593 and DOM15-26-501 weremeasured after i.v. administration DOM15-26-593 and DOM15-26-501 to 3rats at concentrations of 5 mg/kg. Levels of DOM15-26-593 andDOM15-26-501 in the serum were then measured using a direct VEGF bindingstandard ELISA assay and an anti-human Fc antibody, therefore onlyintact drug in the serum samples were detected. The full pharmacokineticprofile is shown in the Table 23 below:

TABLE 23 Summary Pharmacokinetic parameters of the DOM15-26 & DOM15-26-593 Fc fusions in rat Half Life Cmax AUC (0-inf) Clearance DAB ™ (hr)(μg/ml) (hr * μg/ml) (ml/hr/kg) DOM15-26- 12.9 91.4 445.1 11.8 501DOM15-26- 84.6 101.8 3810 1.3 593

It can be seen from these results that DOM15-26-593 has a significantlyimproved pharmacokinetic profile with e.g. an extended half life andreduce clearance rate.

The significantly improved potency and pharmacokinetic properties of theDOM15-26-593 resulted in analysis of the compound for a range of otherbiophysical attributes.

Solution State Properties: Analysis by SEC-MALLs & AUC:

Experiments were Done with DOM15-26-593 as Follows:

DOM15-26-593 behaves as a monomer in solution at concentrations of up to2.5 mg/ml with a calculated molecular mass of 78-81 KDa, consistent withthe calculated intact molecular mass of approx 76 kDa (FIGS. 42a & 42b).

Thermal Melting Properties: Analysis by DSC

Experiments were Done with DOM15-26-593 as Follows:

The increased thermal stability of the trypsin selected DAB™ (65° C.,FIG. 43 middle panel) is maintained in the Fc fusion (64.5° C., FIG. 43upper panel). The Tm curve of the DOM15-26-501 DAB™ (52° C., FIG. 43lower panel) is shown for comparison.

Stability to Freeze-Thaw, Temperature Stress and Serum Components

Experiments were Done with DOM15-26-593 as Follows:

The stability properties of the DOM15-26-593 DAB™ mean that theDOM15-26-593 DAB™ can be subjected to physical and biological stresswith minimal effects on its ability to bind VEGF (FIGS. 44-47 (a andb)). For example, the molecule can be repeatedly freeze thawed fromliquid nitrogen (−196° C.) to body temperature (37° C.) for 10 cycleswithout loss of binding activity as determined by ELISA (FIG. 44). Thistreatment also resulted in no obvious alterations in the molecule'saggregation state, as assessed by conventional size exclusionchromatography (FIG. 45). Further tests demonstrated that the moleculecan be placed at a range of different temperatures from −80° C. to 55°C. with only a minor drop in antigen binding activity after 168 hours atonly the highest incubation temperature (FIG. 46). Furthermore,incubation with serum from human or cynomolgus monkeys at 37° C. for 14days caused no loss of antigen binding ability (FIGS. 47a and 47b ), asdetermined by the VEGF binding ELISA

Potency in VEGFR2Receptor Binding Assay & HUVEC Cell Assay:

The Receptor Binding Assays Described Above were Carried Out as Follows:

The receptor binding assay described above was used to assess thepotency of the Fc fusions (FIG. 48). It was found that the DOM15-26-593DAB™ has enhanced potency in this assay, which establishes the abilityof the DAB™ to block the binding of VEGF to VEGFR2 in vitro. The potencyof the DMS 1529 was also demonstrated in a HUVEC (Human Umbilical VeinEndothelial Cell) assay, where the ability of VEGF antagonists to blockthe VEGF stimulated proliferation of HUVEC cells is measured. Cellnumbers are determined at the end of a fixed incubation period with apredetermined amount of VEGF and a varying amount of test article. Themore potent the antagonist, the lower the cell proliferation observed(FIG. 49).

The nucleotide sequence of DOM1h-131-511 is set out in this paragraph.

The Nucleotide Sequence of DOM1h-131-511:

GAGGTGCAGC TGTTGGAGTC TGGGGGAGGC TTGGTACAGC CTGGGGGGTC CCTGCGTCTC(SEQ ID NO: 262)TCCTGTGCAG CCTCCGGATT CACCTTTGCG CATGAGACGA TGGTGTGGGT CCGCCAGGCTCCAGGGAAGG GTCTAGAGTG GGTCTCACAT ATTCCCCCGG TTGGTCAGGA TCCCTTCTACGCAGACTCCG TGAAGGGCCG GTTCACCATC TCCCGCGACA ATTCCAAGAA CACGCTATATCTGCAAATGA ACAGCCTGCG TGCCGAGGAC ACAGCGGTAT ATTACTGTGC GCTGCTTCCTAAGAGGGGGC CTTGGTTTGA CTACTGGGGT CAGGGAACCC TGGTCACCGT CTCGAGC

The material in the ASCII text file named“DB00056C1SeqList1April2013.txt [,]” created on Apr. 1, 2013 and havinga size of 194,584 bytes is incorporated herein by reference in itsentirety.

The invention claimed is:
 1. A pulmonary formulation comprising animmunoglobulin single variable domain that comprises the amino acidsequence shown in SEQ ID NO: 224, in a particle having a size of 6 μm orless.
 2. The pulmonary formulation of claim 1 having a pH between 6.5and 8.0.
 3. The pulmonary formulation of claim 2 further comprising apolyethylene glycol.
 4. The pulmonary formulation of claim 2 furthercomprising sucrose.
 5. A delivery device comprising the formulation ofclaim 1, wherein said device is selected from the group consisting of aninhaler and an intranasal delivery device.
 6. A delivery devicecomprising the formulation of claim 1, wherein said device is anebulizer.
 7. A delivery device comprising the formulation of claim 2,wherein said device is selected from the group consisting of an inhalerand an intranasal delivery device.
 8. A delivery device comprising theformulation of claim 2, wherein said device is a nebulizer.
 9. Adelivery device comprising the formulation of claim 3, wherein saiddevice is selected from the group consisting of an inhaler and anintranasal delivery device.
 10. A delivery device comprising theformulation of claim 3, wherein said device is a nebulizer.
 11. Adelivery device comprising the formulation of claim 4, wherein saiddevice is selected from the group consisting of an inhaler and anintranasal delivery device.
 12. A delivery device comprising theformulation of claim 4, wherein said device is a nebulizer.
 13. Apulmonary formulation comprising a polypeptide that comprises the aminoacid sequence shown in SEQ ID NO: 224, in a particle having a size of 6μm or less.
 14. The pulmonary formulation of claim 13 having a pHbetween 6.5 and 8.0.
 15. The pulmonary formulation of claim 14 furthercomprising a polyethylene glycol.
 16. The pulmonary formulation of claim14 further comprising sucrose.
 17. A delivery device comprising theformulation of claim 13, wherein said device is selected from the groupconsisting of an inhaler and an intranasal delivery device.
 18. Adelivery device comprising the formulation of claim 13, wherein saiddevice is a nebulizer.
 19. A delivery device comprising the formulationof claim 14, wherein said device is selected from the group consistingof an inhaler and an intranasal delivery device.
 20. A delivery devicecomprising the formulation of claim 14, wherein said device is anebulizer.
 21. A delivery device comprising the formulation of claim 15,wherein said device is selected from the group consisting of an inhalerand an intranasal delivery device.
 22. A delivery device comprising theformulation of claim 15, wherein said device is a nebulizer.
 23. Adelivery device comprising the formulation of claim 16, wherein saiddevice is selected from the group consisting of an inhaler and anintranasal delivery device.
 24. A delivery device comprising theformulation of claim 16, wherein said device is a nebulizer.
 25. Apulmonary formulation comprising an immunoglobulin single variabledomain that comprises the amino acid sequence shown in SEQ ID NO: 224and a pharmaceutically acceptable carrier, in a particle having a sizeof 6 μm or less.
 26. The pulmonary formulation of claim 25 having a pHbetween 6.5 and 8.0.
 27. The pulmonary formulation of claim 26 furthercomprising a polyethylene glycol.
 28. The pulmonary formulation of claim26 further comprising sucrose.
 29. A delivery device comprising theformulation of claim 25, wherein said device is selected from the groupconsisting of an inhaler and an intranasal delivery device.
 30. Adelivery device comprising the formulation of claim 25, wherein saiddevice is a nebulizer.
 31. A delivery device comprising the formulationof claim 26, wherein said device is selected from the group consistingof an inhaler and an intranasal delivery device.
 32. A delivery devicecomprising the formulation of claim 26, wherein said device is anebulizer.
 33. A delivery device comprising the formulation of claim 27,wherein said device is selected from the group consisting of an inhalerand an intranasal delivery device.
 34. A delivery device comprising theformulation of claim 27, wherein said device is a nebulizer.
 35. Adelivery device comprising the formulation of claim 28, wherein saiddevice is selected from the group consisting of an inhaler and anintranasal delivery device.
 36. A delivery device comprising theformulation of claim 28, wherein said device is a nebulizer.
 37. Apulmonary formulation comprising a polypeptide that comprises the aminoacid sequence shown in SEQ ID NO: 224 and a pharmaceutically acceptablecarrier, in a particle having a size of 6 μm or less.
 38. The pulmonaryformulation of claim 37 having a pH between 6.5 and 8.0.
 39. Thepulmonary formulation of claim 38 further comprising a polyethyleneglycol.
 40. The pulmonary formulation of claim 38 further comprisingsucrose.
 41. A delivery device comprising the formulation of claim 37,wherein said device is selected from the group consisting of an inhalerand an intranasal delivery device.
 42. A delivery device comprising theformulation of claim 37, wherein said device is a nebulizer.
 43. Adelivery device comprising the formulation of claim 38, wherein saiddevice is selected from the group consisting of an inhaler and anintranasal delivery device.
 44. A delivery device comprising theformulation of claim 38 that is a nebulizer.
 45. A delivery devicecomprising the formulation of claim 39, wherein said device is selectedfrom the group consisting of an inhaler and an intranasal deliverydevice.
 46. A delivery device comprising the formulation of claim 39,wherein said device is a nebulizer.
 47. A delivery device comprising theformulation of claim 40, wherein said device is selected from the groupconsisting of an inhaler and an intranasal delivery device.
 48. Adelivery device comprising the formulation of claim 40, wherein saiddevice is a nebulizer.